Agonist antibody to human thrombopoietin receptor

ABSTRACT

This invention provides an agonist antibody to a human thrombopoietin receptor (alias: human c-Mpl). More particularly, this invention provides an agonist antibody to a human thrombopoietin receptor, wherein the agonist antibody comprises: antibody constant regions comprising (1) amino acid sequences in a heavy chain constant region and a light chain constant region of a human antibody, (2) an amino acid sequence of a heavy chain constant region with a domain substituted between human antibody subclasses, and an amino acid sequence of a light chain constant region of a human antibody, or (3) amino acid sequences comprising a deletion(s), substitution(s), addition(s), or insertion(s) of one or several amino acid residues in the amino acid sequences of (1) or (2) above; and antibody variable regions capable of binding to and activating a human thrombopoietin receptor; and wherein the agonist antibody has the properties: (a) that the antibody induces colony formation at a concentration of 10,000 ng/ml or lower as determined by the CFU-MK colony formation assay using human umbilical-cord-blood-derived CD34+ cells; and (b) that the antibody has a maximal activity at least 50% higher than that of PEG-rHuMGDF and an 50% effective concentration (EC50) of 100 nM or less in the cell proliferation assay using UT7/TPO cell. Also provided is a pharmaceutical composition for treating thrombocytopenia comprising said antibody.

TECHNICAL FIELD

The present invention relates to an agonist antibody to a humanthrombopoietin receptor (alias: human c-Mpl).

The present invention further relates to a therapeutic agent used for apatient/disease, in cases where there is a clinical need of increasingplatelets, comprising, as an active ingredient, said anti-human c-Mplagonist antibody, particularly a therapeutic agent for thrombocytopenia.

BACKGROUND OF THE INVENTION

<TPO and TPO Receptor>

Thrombopoietin (TPO) is a hematopoietic factor that promotesproliferation of megakaryocytes and platelets in vivo. Human TPO is aglycoprotein comprising 332 amino acid residues in full length, and theN-terminal sequence is known to be important for the activity of humanTPO. Human TPO exhibits its functions upon binding to TPO receptor oncell membrane.

c-Mpl is the only TPO receptor that is known at present. Human c-Mpl isa glycoprotein having one transmembrane domain, comprising 635 aminoacids if it contains signal peptide or 610 amino acids if it is matured,and it belongs to the type I cytokine receptor family. The messenger RNAand protein sequences of human c-Mpl have been already reported(Genbank: NM_005373, NP_005364). Examples of molecules of the samefamily include erythropoietin receptor (EpoR), G-CSF receptor (G-CSFR),and interleukin 3 receptor (IL-3R). Human c-Mpl has 2 CRH (cytokinereceptor homologue) domains in its extracellular region (referred to asCRH1 and CRH2 from the N-terminus), and such domains comprise WSXWS (SEQID NO: 95) motif peculiar to the cytokine receptor family. Theintracellular domain contains 2 sequences, Box1 and Box2, which areessential for signal transduction. It is suggested that TPO binds toCRH1 and dimerizes c-Mpl, thereby transducing a signal; however,specific modes of the binding and activation have not yet beenelucidated. Upon dimerization of c-Mpl, signaling kinase that has boundto the intracellular domain is activated, and phosphorylation signal istransmitted within the cell. It is known that the TPO-Mpl signalactivates Jak-STAT, PI3K-Akt, and Ras-MAPK pathways. In case of a mousein which TPO or c-Mpl is defective, it is reported that the plateletcount decreases to approximately 10%-20% relative to that of a wild-typemouse, indicating that the TPO-Mpl system is a critical system forregulating the platelet counts. c-Mpl expression is observed not only inmegakaryocytes but also in undifferentiated hematopoietic progenitorcells or hematopoietic stem cells. c-Mpl-positive cell fractions in thebone marrow are known to have a higher ability to reconstruct bonemarrow than c-Mpl-negative fractions. It is also known that ac-Mpl-deficient mouse has a decreased number of hematopoietic stemcells, as well as a decreased number of megakaryocytes and platelets(Hiroshi Miyazaki, “Future Prospects for Thrombopoietin,” JapaneseJournal of Transfusion Medicine, 46(3) , 311-316, 2000; and Murone, M.et al., Stem Cell 16: 1-6, 1998). These findings suggest the involvementof the TPO-Mpl system with the hematopoietic system at the stem celllevel or thereafter.

Since the cloning of TPO, its use as a therapeutic agent forthrombocytopenia has been expected, and clinical trials have beenconducted in the past with respect to two types of recombinant TPOs:full-length human TPO (rhTPO) and PEG-rHuMGDF (pegylated recombinanthuman megakaryocyte proliferation and development factor) comprising apegylated peptide sequence of the N-terminal 163 amino acids which formthe active site of human TPO (Kuter, D J et al., Blood 100 (10):3457-69, 2002). In the clinical trails, the recombinant TPOs were foundto successfully increase platelets of healthy volunteers and patientswith idiopathic thrombocytopenic purpura (ITP). Also, effects ofreducing thrombocytopenia caused by nonmyeloablative chemotherapy havebeen demonstrated. Although the number of cases is small, effects ofrecombinant TPOs on patients with aplastic anemia (AA) ormyelodysplastic syndrome (MDS) have been reported (Yonemura, Y. et al.,Int J Hemat (82) 307-309, 2005; and Komatsu, N. et al., Blood 96, 296a,2000).

<c-Mpl Agonist Antibody>

A variety of TPO mimetics having c-Mpl-mediating signaling properties asin TPO but having completely different molecular properties have beenstudied (Broudy, V C et al., Cytokine 25(2): 52-60, 2004; and Wang B. etal., Clin Pharmacol Ther., 76(6): 628-38, 2004). Known mimetics areroughly classified into, for example, peptidic lower molecules,nonpeptidic lower moleculest, antibody-derived molecules, agonistantibodies, and the like.

Examples of known anti-c-Mpl agonist human antibodies include 12B5,12E10, and 12D5 (WO 99/10494). Such antibodies do not have activityagainst primary human cells in the form of a whole antibody, such aswhole IgG. The term “primary human cell” as used herein refers to a cellon which TPO acts in vivo, such as CD34+ cell derived from humanumbilical cord blood or bone marrow, but not an especially establishedcell line which is highly sensitive to TPO or a cell into which TPOreceptor gene has been introduced and expressed at a high level. Theterm refers to cells on which TPO act in vivo, such as CD34+ cellsderived from human umbilical cord blood or bone marrow.

Examples of known murine agonist antibodies include BAH-1 (WO 99/03495;and Deng B. et al., Blood 92(6): 1981-1988, 1998) and VB22B (WO2005/056604). Murine antibodies are known to exhibit antigenicity in thehuman blood and thus are not appropriate as pharmaceuticals. In general,it is difficult to humanize agonist antibodies in the form of a wholeantibody using, for example, CDR grafting while maintaining activity (WO2005/056604; and Ji Hee Son et al., Journal of Immunological Methods286: 187-201, 2004). Even if such known agonist antibodies are presentaccordingly, it is not easy to create agonist human antibodies that acton primary cells.

Antibody-derived lower molecules as described above in relation to theTPO mimetics are also represent a certain type of agonist antibody.Diabody and single chain (Fv)₂ (sc(Fv)₂) that are prepared by modifyingpart of an antibody have been reported (WO 99/10494; and WO2005/056604). The modified antibodies that had been produced by suchtechnique, however, may disadvantageously have antigenecity resultingfrom drastic modification of molecules. Also, their half-lives in bloodwould be shorter than that of the whole antibody. Thus, use of suchmodified antibodies as pharmaceuticals remains problematic.

Thus, the whole antibody has properties useful for pharmaceuticals, suchas low antigenecity or half-life duration in blood; however, it is noteasy to create agonist human antibodies having sufficient activity inthe form of a whole antibody, as described above.

Accordingly, the present inventors have attempted to obtain agonisthuman human antibodies having sufficient activity without drasticallymodifying the antibody structure and, as a result, the present inventorshave now succeeded in obtaining the antibodies of interest as describedbelow. Moreover, the present inventors have now succeeded in modifyingthe hinge region of an antibody, thereby improving an agonist activity.Antibodies produced according to the present invention will be suitableas a therapeutic agent of thrombocytopenia.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a novel anti-humanc-Mpl agonist antibody.

In the present invention, the “antibody” is capable of transducing asignal that is substantially equivalent to that of a natural ligand,TPO, to human c-Mpl (where the signal transducing had been difficult toachieve with a whole antibody) and has a proliferation stimulatingactivity on primary human cells.

The second object of the present invention is to provide a technique forimproving the activity of agonist antibodies without antibodyfragmentation, thus providing a novel anti-human c-Mpl agonist antibodyhaving properties desirable for pharmaceuticals, such as long half-lifeand low antigenecity, that antibody molecules should originally bear.

In order to attain the above objects, the present inventors haveconducted concentrated studies on the anti-human c-Mpl agonist antibody.As a result, the present inventors succeeded in obtaining humanantibodies producing a signal substantially equivalent to that producedby a natural ligand and having activity on human primary cells in theform of a whole antibody. Furthermore, the present inventors haveconducted concentrated studies on the obtained agonist antibodies. As aresult, they have now found a modification technique for improving theagonist activity of an antibody without causing fragmentation thereof.This has led to the completion of the present invention.

Specifically, the present invention comprises the following features.

1. Agonist Antibody to Human Thrombopoietin Receptor

The agonist antibody to human thrombopoietin receptor according to thepresent invention includes the following antibodies (1) to (6).

(1) An agonist antibody to human thrombopoietin receptor, wherein theantibody comprises an antibody constant region comprising any one of thefollowing amino acid sequences (i) to (iii):

(i) amino acid sequences of a heavy chain constant region and a lightchain constant region of a human antibody,

(ii) an amino acid sequence of a heavy chain constant region with adomain substituted between human antibody subclasses and an amino acidsequence of a light chain constant region of a human antibody, or

(iii) an amino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues inthe amino acid sequence (i) or (ii); and

an antibody variable region which is capable of binding to andactivating the human thrombopoietin receptor; and

wherein the antibody has the following properties (a) and/or (b):

(a) that the antibody induces colony formation at a concentration of10,000 ng/ml or lower as determined by the CFU-MK colony formation assayusing human umbilical cord blood derived CD34+ cells; and/or

(b) that the antibody has an activity at least 50% higher than that ofPEG-rHuMGDF, whose structure is described below, and a 50% effectiveconcentration (EC50) of 100 nM or less in the cell proliferation assayusing UT7/TPO cell.

As used herein, the human antibody subclasses include IgG1, IgG2, IgG3,and IgG4. Sequences of a human immunoglobulin constant region or thelike can be obtained from, for example, the NCBI website (e.g., GenBankor UniGene). Examples include Accession No: J00228 for the human IgG1heavy chain constant region, Accession No: J00230 for the human IgG2heavy chain constant region, Accession No: X03604 for the human IgG3heavy chain constant region, Accession No: K01316 for the human IgG4heavy chain constant region, Accession Nos: V00557, X64135, and X64133for the human light chain κ constant region, and Accession Nos: X64132and X64134 for the human light chain λ constant region.

The term “assay for CFU-MK colony formation using human umbilical cordblood derived CD34+ cells” as used herein refers to an assay techniqueas described in Example 6 below, and the antibody concentration requiredfor colony formation can be determined based on said assay technique.

The term “cell proliferation assay using UT7/TPO cell” as used hereinrefers to an assay technique as described in Example 5 below, and theproliferation activity and EC50 can be determined based on said assaytechnique.

The term “PEG-rHuMGDF” as used herein refers to a molecule comprisingthe amino acid sequence as shown in SEQ ID NO: 1, which molecule isprepared by extracting polypeptides produced with the use of E. colithat has been transformed with a plasmid comprising cDNA encoding atruncated protein comprising the amino-terminal receptor binding domainof human TPO (Ulich et al., Blood 86: 971-976, 1995), refolding andpurifying thepolypeptide, and covalently binding the polyethylene glycol(PEG) portion to the amino terminus thereof, this molecule having thefollowing structure:

PEG-NH-SPAPPACDLRVLSKLLRDSHVLHSRLSQCPEVHPLPTPVLLPAVDFSLGEWKTQMEETKAQDILGAVTLLLEGVMAARGQLGPTCLSSLLGQLSGQVRLLLGALQSLLGTQLPPQGRTTAHKDPNAIFLSFQHLLRGKVRFLMLVGGSTLCVRRAPPTTAVPS-COOH.

As used herein, the term “activation of human c-Mpl” refers tointracellularly transducing a human c-Mpl-associated signal in humanc-Mpl-expressing cells.

The term “several” as used herein refers to an integer of, for example,2 to about 10, such as 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4,or 2 to 3.

(2) The antibody according to (1) above having an activity of inducingcolony formation at a concentration of 10,000 ng/ml or lower, preferably1,000 ng/ml or lower, and more preferably 100 ng/ml or lower, amongantibodies having an activity of inducing colony formation as determinedby the colony formation assay and/or a cell proliferation activity asdetermined by the cell proliferation assay using UT7/TPO cell.

(3) The antibody according to (1) above having a cell proliferationactivity at least 50%, preferably at least 70%, and more preferably atleast 90% higher than that of PEG-rHuMGDF, and a 50% effectiveconcentration (EC50) of 100 nM or less, preferably 10 nM or less, andmore preferably 1 nM or less.

(4) The antibody according to (1) above, which exhibits activitiesdescribed below as determined both by the colony formation assay and bythe cell proliferation assay:

(i) An agonist antibody to human thrombopoietin receptor having theproperties (a) and (b) below:

(a) that the antibody induces colony formation at a concentration of10,000 ng/ml or lower as determined by the CFU-MK colony formation assayusing human umbilical cord blood derived CD34+ cells; and

(b) that the antibody has a maximal activity 50% higher than that ofPEG-rHuMGDF having the structure shown below and a 50% effectiveconcentration (EC50) of 100 nM or less in the cell proliferation assayusing UT7/TPO cell.

(ii) An agonist antibody to human c-Mpl having the properties (a) and(b) below:

(a) that the antibody induces colony formation at a concentration of1,000 ng/ml or lower as determined by the CFU-MK colony formation assayusing human umbilical cord blood derived CD34+ cells; and

(b) that the antibody has a maximal activity 70% higher than that ofPEG-rHuMGDF having the structure shown below and an EC50 of 10 nM orless in the cell proliferation assay using UT7/TPO cell.

(iii) An agonist antibody to human c-Mpl having the properties (a) and(b) below:

(a) that the antibody induces colony formation at a concentration of 100ng/ml or lower as determined by the CFU-MK colony formation assay usinghuman umbilical cord blood derived CD34+ cells; and

(b) that the antibody has a maximal activity 90% higher than that ofPEG-rHuMGDF having the structure shown below and an EC50 of 1 nM or lessin the cell proliferation assay using UT7/TPO cell.

(5) The antibody according to (1) above comprising an amino acidsequence of the variable region of the heavy chain and an amino acidsequence of the variable region of the light chain, wherein the aminoacid sequences is selected from the group consisting of the followingamino acid sequences (a) to (h) (where the names described in theparentheses indicate the antibodies described in the Examples below fromwhich the sequences of variable regions are derived):

(a) a heavy chain variable region comprising the amino acid sequence asshown in SEQ ID NO: 2 and a light chain variable region comprising theamino acid sequence as shown in SEQ ID NO: 3 (name of antibody: 7-10);

(b) a heavy chain variable region comprising the amino acid sequence asshown in SEQ ID NO: 4 and a light chain variable region comprising theamino acid sequence as shown in SEQ ID NO: 5 (name of antibody: 4-49);

(c) a heavy chain variable region comprising the amino acid sequence asshown in SEQ ID NO: 6 and a light chain variable region comprising theamino acid sequence as shown in SEQ ID NO: 7 (name of antibody: 6-4-50);

(d) a heavy chain variable region comprising the amino acid sequence asshown in SEQ ID NO: 8 and a light chain variable region comprising theamino acid sequence as shown in SEQ ID NO: 9 (name of antibody: 6-5-2);

(e) a heavy chain variable region comprising the amino acid sequence asshown in SEQ ID NO: 2, and a light chain variable region comprising anamino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues ofthe framework region in the amino acid sequence as shown in SEQ ID NO:3;

(f) a heavy chain variable region comprising the amino acid sequence asshown in SEQ ID NO: 4, and a light chain variable region comprising anamino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues ofthe framework region in the amino acid sequence as shown in SEQ ID NO:5;

(g) a heavy chain variable region comprising the amino acid sequence asshown in SEQ ID NO: 6, and a light chain variable region comprising anamino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues ofthe framework region in the amino acid sequence as shown in SEQ ID NO:7; and

(h) a heavy chain variable region comprising the amino acid sequence asshown in SEQ ID NO: 8, and a light chain variable region comprising anamino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues ofthe framework region in the amino acid sequence as shown in SEQ ID NO:9.

(6) The antibody according to any of (1) to (5) above, wherein theagonist antibody to human c-Mpl is a human antibody.

2. Heavy-Chain-Modified Agonist Antibodies

The heavy chain modified agonist antibodies according to the presentinvention include the following antibodies.

(1) An agonist antibody, wherein the upper hinge region of the heavychain constant region comprises any one of the following amino acidsequences (a) and (b):

(a) the amino acid sequence as shown in SEQ ID NO: 10; or

(b) the amino acid sequence as shown in SEQ ID NO: 11, and

wherein the region of from a middle hinge region to the C terminuscomprises an amino acid sequence of human immunoglobulin G4, or an aminoacid sequence with a mutation of a region associated with propertiesthat are not preferable for agonist antibody concerningantibody-dependent cellular cytotoxicity (ADCC) activity or the like inthe amino acid sequence of human immunoglobulin G4.

The term “upper hinge” as used herein refers to the N-terminal sequencefrom position 216 to position 226 according to the Kabat EU numberingsystem (Kabat et al., Sequences of Proteins of Immunological Interest,5^(th) Ed. Public Health Service, National Institute of Health,Bethesda, Md., 1991). The term “middle hinge” refers to the N-terminalsequence from position 226 to position 231 according to the Kabat EUnumbering system. FIG. 4B shows an amino acid sequence of the upperhinge portion, an amino acid sequence of the middle hinge portion, andamino acid sequences before and after said porions, with respect to therespective subtypes including human immunoglobulin G4. In this figure,CH1 indicates a portion of the CH1 region adjacent to the upper hinge,and CH2 indicates a portion referred to as a lower hinge in the CH2region.

(2) An antibody comprising a heavy chain wherein the region of from themiddle hinge region to the C terminus of the heavy chain constant regioncomprises an amino acid sequence comprising substitutions of serine atposition 228 with proline and leucine at position 235 with glutamic acidin the amino acid sequence of human immunoglobulin G4, where saidpositions are based on the Kabat EU numbering system.

(3) The heavy chain modified antibody according to (2) above, which isan agonist human antibody to human c-Mpl shown in (i) or (ii) below.

(i) An agonist antibody to human c-Mpl comprising a heavy chain whereinthe upper hinge region of the heavy chain constant region comprises anyone of the amino acid sequences (a) or (b):

(a) the amino acid sequence as shown in SEQ ID NO: 10; or

(b) the amino acid sequence as shown in SEQ ID NO: 11, and

wherein the region of from the middle hinge region to the C terminus ofthe heavy chain constant region comprises the amino acid sequence ofhuman immunoglobulin G4 or an amino acid sequence comprisingsubstitutions of serine at position 228 with proline and leucine atposition 235 with glutamic acid in the amino acid sequence of humanimmunoglobulin G4, where said positions are based on the Kabat EUnumbering system.

(ii) According to more preferable embodiment, the agonist antibody tohuman c-Mpl according to (i) above is selected from the group consistingof the antibodies (a) to (h):

(a) an antibody comprising a heavy chain comprising the amino acidsequence as shown in SEQ ID NO: 2 and a light chain comprising the aminoacid sequence as shown in SEQ ID NO: 3;

(b) an antibody comprising a heavy chain comprising the amino acidsequence as shown in SEQ ID NO: 4 and a light chain comprising the aminoacid sequence as shown in SEQ ID NO: 5;

(c) an antibody comprising a heavy chain comprising the amino acidsequence as shown in SEQ ID NO: 6 and a light chain comprising the aminoacid sequence as shown in SEQ ID NO: 7;

(d) an antibody comprising a heavy chain comprising the amino acidsequence as shown in SEQ ID NO: 8 and a light chain comprising the aminoacid sequence as shown in SEQ ID NO: 9;

(e) an antibody comprising a heavy chain comprising the amino acidsequence as shown in SEQ ID NO: 2, and a light chain comprising an aminoacid sequence comprising a deletion(s), substitution(s), addition(s), orinsertion(s) of one or several amino acid residues of the frameworkregion in the amino acid sequence as shown in SEQ ID NO: 3;

(f) an antibody comprising a heavy chain comprising the amino acidsequence as shown in SEQ ID NO: 4 and a light chain comprising an aminoacid sequence comprising a deletion(s), substitution(s), addition(s), orinsertion(s) of one or several amino acid residues of the frameworkregion in the amino acid sequence as shown in SEQ ID NO: 5;

(g) an antibody comprising a heavy chain comprising the amino acidsequence as shown in SEQ ID NO: 6 and a light chain comprising an aminoacid sequence comprising a deletion(s), substitution(s), addition(s), orinsertion(s) of one or several amino acid residues of the frameworkregion in the amino acid sequence as shown in SEQ ID NO: 7; and

(h) an antibody comprising a heavy chain comprising the amino acidsequence as shown in SEQ ID NO: 8 and a light chain comprising an aminoacid sequence comprising a deletion(s), substitution(s), addition(s), orinsertion(s) of one or several amino acid residues of the frameworkregion in the amino acid sequence as shown in SEQ ID NO: 9.

3. Pharmaceutical Use and Pharmaceutical Composition of Agonist Antibodyto Human c-Mpl

The agonist antibody to human c-Mpl according to the present inventionis capable of binding to and activating a c-Mpl receptor, and/or iscapable of stimulating the production of platelets (i.e., the activityof platelet generation) and platelet progenitors (i.e., the activity ofblood megakaryocyte generation) in vivo and in vitro.

Specific examples of the pharmaceutical composition comprising, as anactive ingredient, an agonist antibody to human c-Mpl according to thepresent invention, and the pharmaceutical use thereof include.

(1) A pharmaceutical composition comprising, as an active ingredient,any of the antibodies described in section 1, (1) to (6) and section 2,(3) above.

(2) An agent for increasing platelets comprising, as an activeingredient, any of the antibodies described in section 1, (1) to (6)above and section 2, (3) above.

(3) The agent for increasing platelets according to (2) above, which isused for promoting platelet recovery when bone marrow transplantation orumbilical cord blood transplantation is carried out.

(4) A therapeutic agent for thrombocytopenia comprising, as an activeingredient, any of the antibodies described in section 1, (1) to (6) andsection 2, (3) above.

(5) The therapeutic agent for thrombocytopenia according to (4) above,wherein the thrombocytopenia is any one of the diseases (a) to (f)below:

(a) idiopathic thrombocytopenic purpura (ITP);

(b) thrombocytopenia after cancer chemotherapy;

(c) aplastic anemia;

(d) osteomyelodysplasia syndrome (MDS);

(e) thrombocytopenia attributable to hepatic diseases; or

(f) thrombocytopenia after bone marrow transplantation or umbilical cordblood transplantation.

(6) An agent for increasing blood cells comprising, as an activeingredient, a human c-Mpl agonist antibody used for promoting blood cellrecovery after hematopoietic stem cell transplantation.

(7) The agent for increasing blood cells according to (6) above,comprising, as an active ingredient, any of the antibodies described insection 1, (1) to (6) and section 2, (3) above.

4. Method for Producing Antibody of the Present Invention

The antibodies of the present invention may be produced with the use ofhybridomas that produce the antibodies of the present invention.Alternatively, genes encoding monoclonal antibodies may be cloned fromantibody-producing cells such as hybridomas, and the cloned genes may beincorporated into adequate vectors to produce recombinant antibodiesusing genetic recombination techniques. Preferable examples of methodsfor producing the antibodies of the present invention include methods asset forth below.

A method for producing an agonist antibody to human c-Mpl comprisingpreparing a mammalian animal cell carrying a DNA comprising a heavychain coding nucleotide sequence and a DNA comprising a light chaincoding nucleotide sequence, wherein the nucleotide sequences areselected from the group consisting of (a) to (h) below, and one or moreDNAs comprising a nucleotide sequence that controls the expression ofsaid DNAs, culturing the mammalian animal cell, and isolating andpurifying the expression product of the DNA that encodes an antibodycomprising said heavy chain and light chain from the culture medium inwhich said cell was cultured:

(a) a nucleotide sequence encoding a heavy chain comprising the aminoacid sequence as shown in SEQ ID NO: 2, and a nucleotide sequenceencoding a light chain comprising the amino acid sequence as shown inSEQ ID NO: 3;

(b) a nucleotide sequence encoding a heavy chain comprising the aminoacid sequence as shown in SEQ ID NO: 4, and a nucleotide sequenceencoding a light chain comprising the amino acid sequence as shown inSEQ ID NO: 5;

(c) a nucleotide sequence encoding a heavy chain comprising the aminoacid sequence as shown in SEQ ID NO: 6, and a nucleotide sequenceencoding a light chain comprising the amino acid sequence as shown inSEQ ID NO: 7;

(d) a nucleotide sequence encoding a heavy chain comprising the aminoacid sequence as shown in SEQ ID NO: 8, and a nucleotide sequenceencoding a light chain comprising the amino acid sequence as shown inSEQ ID NO: 9;

(e) a nucleotide sequence encoding a heavy chain comprising the aminoacid sequence as shown in SEQ ID NO: 2, and a nucleotide sequenceencoding a light chain comprising an amino acid sequence comprising adeletion(s), substitution(s), addition(s), or insertion(s) of one orseveral amino acid residues of the framework region in the amino acidsequence as shown in SEQ ID NO: 3;

(f) a nucleotide sequence encoding a heavy chain comprising the aminoacid sequence as shown in SEQ ID NO: 4, and a nucleotide sequenceencoding a light chain comprising an amino acid sequence comprising adeletion(s), substitution(s), addition(s), or insertion(s) of one orseveral amino acid residues of the framework region in the amino acidsequence as shown in SEQ ID NO: 5;

(g) a nucleotide sequence encoding a heavy chain comprising the aminoacid sequence as shown in SEQ ID NO: 6, and a nucleotide sequenceencoding a light chain comprising an amino acid sequence comprising adeletion(s), substitution(s), addition(s), or insertion(s) of one orseveral amino acid residues of the framework region in the amino acidsequence as shown in SEQ ID NO: 7; and

(h) a nucleotide sequence encoding a heavy chain comprising the aminoacid sequence as shown in SEQ ID NO: 8, and a nucleotide sequenceencoding a light chain comprising an amino acid sequence comprising adeletion(s), substitution(s), addition(s), or insertion(s) of one orseveral amino acid residues of the framework region in the amino acidsequence as shown in SEQ ID NO: 9.

5. DNA of the Present Invention

Examples of DNAs of the present invention are set forth below.

(1) Novel DNA comprising a nucleotide sequence that encodes the aminoacid sequence in the heavy chain variable region of an agonist antibodyto human Mpl, and comprising a nucleotide sequence that encodes an aminoacid sequence selected from the group consisting of (a) to (d) below:

(a) the amino acid sequence as shown in SEQ ID NO: 2;

(b) the amino acid sequence as shown in SEQ ID NO: 4;

(c) the amino acid sequence as shown in SEQ ID NO: 6; and

(d) the amino acid sequence as shown in SEQ ID NO: 8.

(2) Novel DNA comprising a nucleotide sequence that encodes the aminoacid sequence in the light chain variable region of an agonist antibodyto human Mpl, and comprising the nucleotide sequence that encodes anamino acid sequence selected from the group consisting of (a) to (h)below:

(a) the amino acid sequence as shown in SEQ ID NO: 3;

(b) the amino acid sequence as shown in SEQ ID NO: 5;

(c) the amino acid sequence as shown in SEQ ID NO: 7;

(d) the amino acid sequence as shown in SEQ ID NO: 9;

(e) an amino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues ofthe framework region in the amino acid sequence as shown in SEQ ID NO:3;

(f) an amino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues ofthe framework region in the amino acid sequence as shown in SEQ ID NO:5;

(g) an amino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues ofthe framework region in the amino acid sequence as shown in SEQ ID NO:7; and

(h) an amino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues ofthe framework region in the amino acid sequence as shown in SEQ ID NO:9.

(3) DNA according to the (1) or (2) above, which encodes an antibodyheavy chain or light chain comprising a variable region and a constantregion.

(4) DNA encoding the antibody heavy chain according to the (3) above,wherein the upper hinge region of the heavy chain constant region of theantibody comprises any one of the following amino acid sequences (a) and(b):

(a) the amino acid sequence as shown in SEQ ID NO: 10; or

(b) the amino acid sequence as shown in SEQ ID NO: 11, and

wherein the region of from the middle hinge region to the C terminus ofthe heavy chain constant region comprises the amino acid sequence ofhuman immunoglobulin G4 or an amino acid sequence comprisingsubstitutions of serine at position 228 with proline and leucine atposition 235 with glutamic acid in the amino acid sequence of humanimmunoglobulin G4, where said positions are based on the Kabat EUnumbering system:

This description includes all or part of the contents as disclosed inthe specification and/or drawings of Japanese Patent Application No.2006-81322 and No. 2006-299554, which are priority documents of thepresent application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows binding activities of agonist antibodies. Bindingactivities of the indicated antibodies were examined by flow cytometryusing FDCP-hMpl cells and FDCP2 cells (FDCP parent cells) (see Example2). The results show that each antibody binds specifically to humanc-Mpl.

FIG. 2, A to D, shows the results of UT7/TPO assay, specifically theproliferation curves of the purified antibodies (IgG1) in the UT7/TPOcell proliferation assay (see Example 5).

FIG. 3 shows the results of CFU-Mk assay, which are the results ofcolony formation assay using human umbilical cord blood derived CD34+cells (see Example 6).

FIG. 4A shows the structure of the N5KG1 vector associated with thepreparation of a recombinant antibody. “C” represents a cytomegaloviruspromoter/enhancer, “B” represents a bovine proliferation hormonepolyadenylation region, “N1” represents exon 1 of neomycinphosphotransferase, “K” represents a human immunoglobulin κ constantregion, “G1” represents a human immunoglobulin γ1 constant region, “BT”represents a murine β globulin major promoter, “N2” represents the exon2 of neomycin phosphotransferase, “D” represents dihydrofolatereductase, “VH” represents a heavy chain variable region, and “VL”represents a light chain variable region.

FIG. 4B shows amino acid sequences of natural human immunoglobulins, andamino acid sequences of CH1 regions and hinge regions (i.e., upper hingeand middle hinge regions) of IgG4PE, IgG4344, IgG4344h1, IgG4344uh, andIgG4344uhm associated with the preparation of recombinant antibodies.Figure discloses SEQ ID NOS 96-104, respectively, in order ofappearance.

FIG. 4C (i.e., FIG. 4C-1 to FIG. 4C-3) shows a process for preparing theexpression vectors N5KG1_(—)7-10 and N5KG1_(—)4-49 for use in thepreparation of recombinant antibodies.

FIG. 4D (i.e., FIG. 4D-1 to FIG. 4D-3) shows a process for preparing theexpression vectors N5KG1_(—)6-4-50 and N5KG1_(—)6-5-2 for use in thepreparation of recombinant antibodies.

FIG. 4E shows the sequences of the constant regions of differentmodified heavy chains associated with the preparation of recombinantantibodies (SEQ ID NOS 74-80, respectively, in order of appearance).

FIG. 4F (i.e., FIG. 4F-1 and FIG. 4F-2) shows the nucleic acid (SEQ IDNO: 81) and amino acid (SEQ ID NO: 82) sequences of 7-10G4344uhm heavychains associated with the preparation of recombinant antibodies.

FIG. 4G shows the nucleic acid (SEQ ID NO: 83) and amino acid (SEQ IDNO: 84) sequences of 7-10G4344uhm light chains associated with thepreparation of recombinant antibodies.

FIG. 5 shows activities of hinge-modified antibodies. FIG. 5A representsactivities of 4-49G1, 4-49G3311, and 4-49G3331 determined by UT7/TPOcell proliferation assay, and FIG. 5B represents activities of7-10G4344uhm and 4-49G4344uhm as determined by UT7/TPO cellproliferation assay.

FIG. 6A shows the results of signaling analysis concerning the agonistantibodies 7-10G4344uhm and 4-49G4344uhm (see Example 11).

FIG. 6B shows the results of signaling analysis concerning the agonistantibodies 6-5-2G1 and 6-5-2G3344 (see Example 11).

FIG. 7 shows human platelet priming effects, which are the results ofthe test described in Example 12. The priming effect of the agonistantibody 7-10G3311 or 4-49G3311 on human platelets is shown. Also,platelet aggregation did not occur by each agonist antibody only(without ADP).

FIG. 8 is a graph showing changes in platelet counts afteradministration of agonist antibodies to cynomolgus monkeys. As describedin Example 13, the agonist antibodies were administered to cynomolgusmonkeys, and then platelet counts were monitored. Arrows indicate thedates of the first administration (PEG-rHuMGDF) and the secondadministration (agonist antibody).

FIG. 9A shows changes in peripheral human platelet counts over timeafter transplantation of 1,000 CD34+ cells (right panel) or 10,000 CD34+cells (left panel) into NOG umbilical cord blood transplantation mousemodels, followed by administration of an analyte. “Pre” indicatesplatelet counts before administration.

FIG. 9B shows human progenitor cell counts (colony counts; GM+E+GEM) inthe bone marrow 6 weeks after transplantation of 1,000 CD34+ cells(right panel) or 10,000 CD34+ cells (left panel) into NOG umbilical cordblood transplantation mouse models, followed by administration of testsubstances. The “progenitor cell counts” refers to total counts of cellsother than megakaryocytes, “GM” refers to granulocytes and macrophages,“E” refers to erythrocytes, and “GEM” refers tocolony-forming-unit-granulocyte-macrophage-erythroids. The results arerepresented by a mean± standard deviation (mean±SD). “Vehicle”represents PBS (phosphate buffered saline) as a control, and “NT”represents non-treated.

FIG. 9C shows chimeric rates of peripheral human cells 6 weeks aftertransplantation of 1,000 CD34+ cells (right panel) or 10,000 CD34+ cells(left panel) into NOG umbilical cord blood transplantation mouse models,followed by administration of an analyte. “Vehicle” represents PBS(phosphate buffered saline) as a control, and “NT” representsnon-treated.

FIG. 10 shows daily changes in platelet counts after the administrationof agonist antibodies to human Mpl Tg mice. TPO or vehicle (PBS) wasadministered to the Tg mouse as the control, 10 μg of 7-10G4344uhm wasadministered to non-Tg mouse (wild-type; non-Tg), and the results of theexperiments are shown in the figure. The results are indicated by mean±SEM.

FIG. 11 shows the binding of light-chain modified antibodies of theagonist antibody 7-10G4344uhm to FM3A-hMpl cell.

FIG. 12 shows UT-7/TPO cell proliferation assay results oflight-chain-modified antibodies of the agonist antibody 7-10G4344uhm.

BEST MODES FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be described in more detail.

The present invention provides anti-human c-Mpl agonist human antibodiesthat act on primary human cells.

The antibodies of the present invention can be isolated by aconventional method for preparing monoclonal antibodies by immunizinghuman antibody-producing mice (e.g., KM mouse™, Kirin Brewery Co., Ltd.)with human Mpl recombinant proteins or human Mpl-expressing cells.Alternatively, antibody genes may be isolated from hybridomas,expression vectors may be constructed, expressing cells may be prepared,and recombinant antibodies having different constant regions may beprepared during such process.

1. Antibodies of the Present Invention

The term “antibody” as used herein refers to an antibody comprising Fab,hinge and Fc regions. Examples of the antibody includes anaturally-occurring antibody, or an antibody produced by a monoclonalantibody-producing hybridoma obtained by known techniques such that ithas the constitution similar to that of the naturally occurringantibody, an antibody genetically engineered from an antibody gene thatis obtained in advance, and an antibody genetically engineered bypartial modification using site-directed mutagenesis. The agonistantibody to human cMpl and the heavy chain-modified agonist antibody ofthe present invention are as described above.

In general, agonist antibodies bind to target molecules on the cellmembrane to form complexes, thereby transmitting a signal. An agonistantibody to a homodimer-forming cytokine receptor family, such aserythropoietin receptor (EpoR), G-CSF receptor (G-CSFR), orthrombopoietin receptor (c-Mpl), is expected to form a dimer uponbinding of a divalent antibody to two molecules. This is suggested bythe fact that many agonist antibodies having the Fab fragments only donot exhibit the activity.

It is considered important that two antigen-binding sites are easilyapproach each other at the time of complex formation. This is alsosuggested by the fact that an antibody that does not have sufficientactivity in the form of a whole antibody exhibits an elevated agonistactivity when it is converted into a low molecular form, such assc(Fv)₂. However, such a low molecular antibody may raise an issue ofantigenecity due to its considerable modification, and its bloodhalf-life may be shortened. Thus, use of a low molecular antibody as apharmaceutical leaves problems to be solved. In order to practicallyutilize such properties that the whole antibody bears useful as apharmaceutical, such as low antigenecity or blood half-life length, theagonist antibody that has a high activity without significantlymodifying its structure would be desirable.

As described in Example 2 below, the present inventors have now improvedthe conventional immunization method to obtain an anti-human c-Mplagonist antibody with high activity in the form of a whole antibody.Examples of the improved immunization are immunization withhigh-expression cell strains and immunization with constantly activemutant receptor expressing cells. Such agonist antibody is demonstratedthat it induces colony formation by means of a colony assay using thehuman umbilical cord blood derived CD34+ cells as described in Example 6below, suggesting that the antibody is useful as a pharmaceutical.

The present inventors have now further attempted to improve flexibilityof the hinge portion to increase the efficiency of complex formation andto enhance the agonist activity. An example of a highly flexiblesequence is a glycine linker. As an alternative example, a hinge regionof IgG3 having the highest flexibility among human IgGs may also beused. It is desirable that a natural sequence is used in order not toimpaire the low antigenecity of the antibody. Thus, an IgG3 hingesequence is more preferable.

Further, an antibody that has an upper hinge region of human IgG3 as aconstant region optimal for the agonist antibody having low cytotoxicityand high hinge flexibility and that has a human IgG4 sequence as aregion from the middle hinge to the C-terminal end, may be prepared bygenetically engineering modification.

More specifically, an antibody is converted into an antibody of adifferent subclass by the genetically engineering modification wellknown in the art (e.g., see EP314161), i.e., DNA that encodes a variableregion of the antibody of the present invention may be used to modify anantibody into an antibody of a different subclass by genetic engineeringprocedures. Further, serine at position 228 of the constant region ofhuman IgG4 heavy chain, which position is based on the EU numberingsystem (see Sequences of proteins of immunological interest, NIHPublication No. 91-3242), may be varied into proline, therebysuppressing formation of a monomer resulting from intramolecularcrosslinking of IgG4 (an S—S bond). Also, leucine at position 235 may bevaried into glutamic acid, thereby reducing activity ofantibody-dependent cellular cytotoxicity (ADCC). IgG4 comprising such 2mutations is referred to as IgG4PE.

In view of the above, the present inventors have now produced a constantregion that was optimal for an agonist antibody and had low cytotoxicityand high hinge flexibility. This constant region has an upper hingeregion of human IgG3, and the region from the middle hinge to theC-terminal end has a human IgG4 sequence. This constant region may becombined with a variable region of an anti-c-Mpl agonist antibody toproduce an agonist antibody having higher safety and activity.

2. Method for Producing the Antibody of the Present Invention

The antibodies of the present invention can be produced by a variety oftechniques. At the outset, production of a hybridoma is required forproducing the antibody of the present invention. When human antibodiesmay be produced by immunizing mice or other animals with an antigenrelevant to the present invention as described in Example 1 below, inparticular, non-human mammals, such as human antibody-producingtransgenic mice, are immunized, for example. Monoclonal antibodies canbe obtained in accordance with conventional techniques, i.e., byallowing antibody-producing cells obtained from a sensitized animal tofuse with a myeloma cell having no capacity of producing autoantibodies,thereby obtaining hybridomas, culturing the hybridomas, and selectingclones that produce monoclonal antibodies exhibiting specific affinitywith the antigen used for immunization. It is required that agonistantibodies are further selected from among the obtained antibodies,which may be carried out by a method established as a technique forassaying activity of a ligand that reacts with a target receptor of anagonist antibody. An agonist antibody to human c-Mpl can be adequatelyselected by a method established as a method for assaying TPO activity,such as UT7/ TPO cell proliferation assay as described in Example 5below.

Production of the agonist antibody to human c-Mpl of the presentinvention, and particularly the monoclonal antibody, involves thefollowing steps. That is, (1) purification of biopolymers used as theimmunogen and/or preparation of cells comprising antigen proteinsoverexpressed on the cell surface; (2) immunization of an animal byinjection of an antigen, blood sampling, assay of an antibody titer, anddetermination of the timing of extraction of the spleen or the like,followed by preparation of antibody-producing cells; (3) preparation ofmyeloma cells; (4) cell fusion between the antibody-producing cell andthe myeloma cell; (5) selection of hybridomas that produce antibodies ofinterest; (6) division into unicellular clones (cloning); (7)optionally, culture of hybridomas for mass-production of monoclonalantibodies, or breeding of animals into which hybridomas have beentransplanted; (8) examination of physiological activity andrecognition/specificity of the thus-produced monoclonal antibodies, orassay for properties as label reagent; (9) cloning of monoclonalantibody genes, and preparation of recombinant antibodies; and the like.

Hereafter, a method for preparing agonist monoclonal antibodies to humanc-Mpl will be described in detail with reference to the above steps,although methods for producing such antibodies are not limited thereto.For example, antibody-producing cells and myeloma cells other thanspleen cells can also be used.

(1) Antigen

When a human c-Mpl antibody is to be obtained, in general, a peptide ischemically synthesized from the c-Mpl amino acid sequence by a methodwell-known in the art since the primary structure of human c-Mpl proteinis known (see GenBank: NP_(—)005364), and the obtained peptide can beused as the antigen. Also, a solubilized c-Mpl recombinant protein thatlacks the transmembrane region and the intramembrane region of c-Mpl canbe used as the antigen.

Alternatively, various human blood megakaryocyte cell lines or humanc-Mpl-expressing cell lines, such as cell lines forcibly expressingc-Mpl, may be used as said antigens. Although various human bloodmegakaryocyte cell lines or forcibly expressed cell lines are known ashuman c-Mpl expressing cell lines, the c-Mpl expression level in suchcell lines is as low as several thousands molecules per cell, and thussuch cell lines are not suitable for the antigen. When a humanantibody-producing mouse (e.g., KM mouse™) is immunized with theexpression cell line FDCP-hMpl comprising human c-Mpl introduced intoFDCP2 (i.e., the murine hematopoietic cell line) (see FEBS Lett. Oct.21, 1996; 395 (2-3): 228-234), increase in the antibody titer is in factinsufficient, and the hMpl-specific human antibody could not beobtained. When the human blood megakaryocyte cell lines are used as theantigens, antibodies that react with other membrane molecules are alsoinduced, so the use of said cell lines is not always suitable forefficiently inducing c-Mpl-specific antibodies. When antigenprotein-expressing cell lines other than human c-Mpl antibodies are usedfor immunization for the purpose of obtaining antibodies having agonistactivity, accordingly, cells that exhibit high expression levels arepreferably selected. Use of murine cell lines, optimally MHC-compatiblecell lines, into which human c-Mpl has been introduced and expressed ata high level, as host cells, is particularly preferable. Examples of themurine cell lines include the cells as described in Example 1 below,i.e., cells obtained by using pEF-MPL635 or pCMV-MPL635 carrying thefull-length human c-Mpl gene as an expression vector and murine L929 orFM3A cell line as a host cell).

Instead of wild-type human c-Mpl, a cell line in which a constantlyactive mutant of human c-Mpl (e.g., a mutant which Trp at position 508has been mutated into Ser and constantly transmits agonist signals in aligand-independent manner; Abe M. et al., Leukemia, August 2002; 16(8):1500-1506) is forcibly expressed in the same manner may be used. Sincesuch mutant is expected to have a three-dimensional structure differentfrom that of a wild-type, an antibody exhibiting high affinity to such aconstantly active mutant may exhibit potent agonist activity.

The above-mentioned forcible expression-exhibiting cell lines can beused as the antigens in optional combination with human cMPL, itsextracellular soluble region, or the like.

(2) Process for Preparing Antibody-Producing Cells

The antigens obtained in (1) above are mixed with the Freund's completeor incomplete adjuvant or an adjuvant such as potash alum, and theresulting mixture is administered to a test animal as the immunogen. Anoptimal test animal is a mouse that is capable of producing a humanantibody via genetic modification (i.e., a human antibody-producingmouse).

The human antibody-producing mouse (e.g., KM mouse™) used in the presentinvention lacks the endogenous mouse immunoglobulin (Ig) heavy chain andthe mouse κ light chain, and such mouse comprises a chromosome 14fragment comprising human Ig heavy chain gene (SC20) and a human Igκchain transgene (KCo5) concurrently. This mouse is prepared by crossinga mouse of lineage A having the human Ig heavy chain locus with a mouseof lineage B having the human Igκ chain transgene. Lineage A is ahomozygote for both the endogenous Ig heavy chain and the destroyed κlight chain and is a mouse lineage carrying an offspring-transmittablechromosome 14 fragment (SC20) (Tomizuka et al., Proc. Natl. Acad. Sci.USA., 2000. Vol. 97: 722). Lineage B is a homozygote for both theendogenous mouse Ig heavy chain and the defective κ light chain and is amouse lineage carrying the human Igκ chain transgene (KCo5) (Nat.Biotechnol., 1996, Vol. 14: 845). Accordingly, the KM mouse is capableof producing a human antibody, which lacks the murine Ig heavy chain andκ chain.

When immunizing a mouse, an immunogen is administered by any ofsubcutaneous injection, intraperitoneal injection, intravenousinjection, endodermic injection, intramuscular injection, or footpadinjection, preferably intraperitoneal injection, footpad injection, orintravenous injection.

Immunization can be carried out once or several times at adequateintervals (preferably at the intervals of 2 to 4 weeks). Thereafter, theantibody titer in the blood serum of the immunized animal against theantigen is assayed, and the animal exhibiting a sufficiently highantibody titer may be used as a source of an antibody-producing cell,whereby the effect of subsequent procedures is enhanced. In general, anantibody-producing cell obtained from an animal 3 to 5 days after finalimmunization is preferably used for subsequent cell fusion.

Examples of methods for assaying the antibody titer that can be usedinclude various known techniques, such as flow cytometory, radioisotopeimmunoassay (hereafter referred to as “the RIA method”), solid phaseenzyme immunoassay (hereafter referred to as “ELISA”), fluorescentantibody method, and passive haemagglutination. From the viewpoint ofdetection sensitivity, promptness, accuracy, or the possibility ofautomation of the procedure, for example, flow cytometory or ELISA ismore preferable.

In the present invention, the antibody titer can be assayed in themanner described below, for example, in the case of flow cytometory.First, the antigen-expressing cells are allowed to react with a humanantibody-containing specimen (e.g., a mouse blood serum, culturesupernatant of hybridoma, or purified antibody). Then, an antibody tohuman antibody, which has been fluorescent-labeled as the secondaryantibody, is added to bind to the human antibody, the resultant iswashed, and the amount of the secondary antibody bound to the cells isassayed by fluorescence. Thus, the antibody titer is determined.

(3) Process for Preparing Myeloma Cells

Cells incapable of producing autoantibodies that are derived frommammalians, such as mice, rats, guinea pigs, hamsters, rabbit, orhumans, can be used as myeloma cells. In general, myeloma cell linesestablished from mice, such as 8-azaguanine resistant mouse(BALB/c)-derived myeloma cell lines P3X63Ag8U.1 (P3-U1) (Yelton, D. E.et al., Current Topics in Microbiology and Immunology, 81, 1-7, 1978),P3/NSI/1-Ag4-1 (NS-1) (Kohler, G. et al., European J. Immunology, 6,511-519, 1976), Sp2/O-Ag14 (SP-2) (Shulman, M. et al., Nature, 276,269-270, 1978), P3X63Ag8.653 (653) (Kearney, J. F. et al., J.Immunology, 123, 1548-1550, 1979), P3X63Ag8 (X63) (Horibata, K. andHarris, A. W. Nature, 256, 495-497, 1975), or the like are preferablyused. Such cell lines are subjected to subculture in an adequate medium,for example, 8-azaguanine medium (i.e., RPMI-1640 medium supplementedwith glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum(hereafter referred to as “FCS”), containing 8-azaguanine), Iscove'smodified Dulbecco's medium (hereafter referred to as “IMDM”), orDulbecco's modified Eagle medium (hereafter referred to as “DMEM”). Saidcell lines are subjected to subculture in a normal medium (e.g., DMEMmedium containing 10% FCS) 3 or 4 days prior to cell fusion in order toensure the number of 2×10⁷ or more cells on the day of cell fusion.

(4) Cell Fusion

Antibody-producing cells are blood plasma cells and progenitor cellsthereof, i.e., lymphocytes. Such cells may be obtained from any sites ofindividuals. In general, antibody-producing cells can be obtained fromspleen cells, lymph nodes, bone marrow, tonsilla, peripheral blood, orany adequate combination thereof Use of spleen cells is the most common.

After the final immunization, a site containing antibody-producingcells, such as spleen, is excised from a mouse that exhibits a givenantibody titer to prepare antibody-producing spleen cells. Subsequently,the spleen cells may be fused to the myeloma cells. At present, the mostcommon technique for allowing the spleen cells to fuse to the myelomacells obtained in step (3) is a method involving the use of polyethyleneglycol, which is relatively low cytotoxicity and provides a simplefusion operation. This method comprises the following procedures, forexample.

The spleen cells and the myeloma cells are thoroughly washed with aserum-free medium (e.g., DMEM) or phosphate buffered saline (hereafterreferred to as “PBS”), the spleen cells are mixed with myeloma cells ata ratio of about 5:1 to 10:1, and the resultant is centrifuged. Thesupernatant is removed, the precipitated cell mass is thoroughlyloosened, and serum-free medium containing 1 ml of 50% (w/v)polyethylene glycol (molecular weight: 1000 to 4000) is added dropwisethereto with stirring. Thereafter, 10 ml serum-free medium is slowlyadded, followed by centrifugation. The supernatant is discarded again,the precipitated cells are suspended in a normal medium containingadequate amounts of hypoxanthine/aminopterin/thymidine (hereafterreferred to as “HAT”) and human interleukin-6 (hereafter referred to as“IL-6”) (hereafter referred to as “HAT medium”), the resultantingsuspension is portioned with each well of a culture plate (hereafterreferred to as “plate”), and culture is then conducted in the presenceof 5% CO₂ at 37° C. for about 2 weeks. During culture, the HAT medium isadequately added.

(5) Selection of Hybridoma

When the above-mentioned myeloma cells are 8-azaguanine resistant celllines, i.e., hypoxanthine/guanine/phosphoribosyltransferase(HGPRT)-deficient cell lines, non-fused myeloma cells andmyeloma/myeloma fused cells cannot survive in HAT-containing medium.Although fused cells between antibody-producing cells or hybridomasbetween antibody-producing cell and myeloma cell can survive, the lifetime of the fused cells between antibody-producing cells is limited. Bycontinuing the culture in HAT-containing medium, accordingly, onlyhybridomas that are fused cells between antibody-producing cell andmyeloma cell survive, leading to selection of hybridomas. The HAT mediumfor hybridomas that have been grown into colonies is exchanged with amedium prepared by removing aminopterin from HAT medium (hereafterreferred to as “HT medium”). Thereafter, part of the culture supernatantis sampled, and the anti-human c-Mpl antibody titer is assayed by, forexample, flow cytometory. A method involving the use of the 8-azaguanineresistant cell lines was described above. It should be noted that othercell lines can also be used depending to methods for selectinghybridomas, and the composition of the medium also varies in such acase.

(6) Cloning Step

The hybridomas that had been found to produce specific antibodies as aresult of the assay of the antibody titer in the same manner as in thecase of (2) above are transferred to another plate and then subjected tocloning. Examples of cloning techniques include: limiting dilutionwherein hybridomas are diluted in such a manner that one hybridoma iscontained per well, and culture is then conducted; a soft agar methodwherein hybridomas are cultured in a soft agar medium and colonies arethen recovered; a method wherein every cell is extracted using amicromanipulator and then cultured; and sorter clone wherein a cell isseparated using a cell sorter. Limiting dilution is simple and oftenemployed.

The wells in which the antibody titer is observed are repeatedlysubjected to cloning 2 to 4 times via, for example, limiting dilution,and the wells in which the antibody titer is stably observed areselected as anti-human c-Mpl monoclonal antibody-producing hybridomalines.

(7) Selection of Agonist Antibodies

The culture supernatant of the obtained anti-human c-Mpl monoclonalantibody-producing hybridoma lines or the antibodies purified from thesupernatant in accordance with procedures as described in (8) below canbe assayed by various TPO activity assay systems to select agonistantibodies. An example of a preferable screening technique is a methodwherein human Mpl is expressed in a mammalian cell and cellproliferation assay is then carried out. For example, proliferationassay (Orita et al., Blood. Jan. 15, 2005; 105(2): 562-6) using thehuman Mpl-expressing BaF3 mouse cell lines may be employed. Since themouse cells may not always reflect the reaction of human cells, it ismore preferable to employ a proliferation assay technique that useshuman Mpl-expressing human cells, in order to select antibodies havingstronger activity on human cells. A specific example of a systeminvolving the use of human cells is a cell proliferation assay using theUT7/TPO cells described in Example 5 below.

(8) Preparation of Monoclonal Antibody by Culturing Hybridoma

The hybridomas that have been cloned are cultured by exchanging the HTmedium with the normal medium. Mass-culture can be performed via rotaryculture using large culture bottles, spinner culture, or culture using ahollow fiber system. The supernatant obtained via such mass-culture canbe purified by a method well-known in the art, such as gel filtration,to obtain anti-human c-Mpl monoclonal antibodies. Alternatively, saidhybridomas may be grown in the abdominal cavity of a mouse of the samelineage (e.g., BALB/c) or a nu/nu mouse, rat, guinea pig, hamster, orrabbit to obtain the ascite containing a large quantity of an anti-humanc-Mpl monoclonal antibody. An example of the technique for simplypurifying monoclonal antibodies is the use of a commercially availablemonoclonal antibody purification kit (e.g., the MAbTrap GII kit;Amersham Pharmacia Biotech). The thus-obtained monoclonal antibodieshave a high antigen specificity to human c-Mpl.

(9) Assay of Monoclonal Antibody

The isotype and the subclass of monoclonal antibodies obtained as abovecan be determined in the following manner. Examples of such techniquesinclude the Ouchterlony method, ELISA, and RIA. Although the Ouchterlonymethod is simple, this technique requires a procedure of concentrationif the concentration of a monoclonal antibody is low. When ELISA or RIAis employed, the culture supernatant as such is allowed to react withthe antigen-adsorbed solid-phase, and secondary antibodies reacting withvarious immunoglobulin isotypes and subclasses are used. Thus, theisotypes and the subclasses of the monoclonal antibodies can beidentified. Furthermore, proteins can be quantified by the Follin-Lowrymethod or based on an absorbance at 280 nm (1.4 (OD280)=immunoglobulin 1mg/ml). Also, monoclonal-antibody-encoding genes can be cloned fromhybridomas to determine the sequences. Thus, the subclass can beidentified.

(10) Cloning of Genes that Encode Monoclonal Antibodies and Preparationof Recombinant Antibodies

Monoclonal antibody-encoding genes are cloned from antibody-producingcells, such as hybridomas, the cloned genes are incorporated intoadequate vectors, and the resultanting vector is introduced into hostcells (e.g., mammalian cell lines, yeast cells, or insect cells). Thus,recombinant antibodies can be prepared via genetic recombinationtechniques (P. J. Delves., Antibody production essential techniques,1997, WILEY, P. Shepherd and C. Dean., Monoclonal Antibodies, 2000OXFORD UNIVERSITY PRESS, J. W. Goding, Monoclonal Antibodies: principlesand practice, 1993, ACADEMIC PRESS).

The present invention includes a nucleic acid comprising a gene sequencefor an antibody carried by the hybridoma that produces the antibody ofthe present invention, and in particular, nucleic acids of a heavy chainvariable region and a light chain variable region of the antibodyproduced by the hybridoma of the present invention. The term “nucleicacid” used herein includes DNA and RNA.

In order to prepare genes encoding monoclonal antibodies fromhybridomas, DNAs each encoding the V region of a L chain, the C regionof a L chain, the V region of a H chain, and the C region of a H chainof the monoclonal antibodies are prepared by PCR or other methods. OligoDNA designed from the antibody gene or amino acid sequence can be usedas a primer, and DNA prepared from a hybridoma can be used as atemplate. Such DNAs are incorporated into an adequate vector, and theresultanting vector is introduced into a host cell for expression.Alternatively, such DNAs are each incorporated into adequate vectors forcoexpression.

A phage or plasmid vector that can autonomously grow in a hostmicroorganism is used. Examples of plasmid DNAs include E. coli-,Bacillus subtilis-, and yeast-derived plasmids. An example of phage DNAis λ phage.

Eukaryotic cells can be used as hosts for transformation since thethree-dimensional structure of the antibody can be accurately formed.Examples thereof include yeast, animal cells such as COS or CHO cells,and insect cells. When animal host cells are used, in particular, aN5KG1-Val Lark vector (IDEC Pharmaceuticals: U.S. Pat. No. 6,001,358)can be used, for example, This vector is an expression vector used forexpressing a recombinant antibody in an animal cell, which comprises twoCMV promoters/enhancers and, downstream thereof, cloning sites of theheavy chain and light chain variable regions. Further, this vectororiginally comprises, downstream of such sites, gene sequences encodingthe constant region of the human γ1 chain and the constant region of thehuman κ chain. Arbitrary heavy chain and light chain variable regionsmay be incorporated into the cloning site of the variable region of thevector in such a manner that reading frames thereof coincide with eachother. Thus, an antibody comprising the light chain variable regionligated to the constant region of the human κ chain and the heavy chainvariable region ligated to the constant region of the human γ1 can beexpressed. An animal cell into which the vector has been introducedproduces an antibody (human IgG1) in a culture solution. Also, a vectorcomprising a gene of a different heavy chain constant region can beused. For example, the N5KG4PE vector (IDEC Pharmaceuticals) comprises,as the gene of the constant region, a sequence comprising the above twomutations (i.e., Ser228Pro and Leu235Glu) introduced into human γ4.Arbitrary gene sequences of heavy chain and light chain variable regionsmay be incorporated into the N5KG4PE vector to express IgG4PE comprisingan arbitrary variable region. Further, the heavy chain or light chaingene may be modified, so that antibodies comprising various constantregions can be prepared.

It should be understood that expression vectors for mammalian cells usedin the present invention are not limited to those described above. Forexample, the other expression vector comprising the CMVpromoter/enhancer as nucleotide sequences for regulating the expressionmay be used. Alternatively, a known promoter(s)/enhancer (s) differentfrom the aforementioned one may be used as an expression-regulatingsequence. Examples of promoters include those obtained from the genomesof viruses, such as polyoma virus, fowlpox virus (UK2211504 published onJul. 5, 1989), adenovirus (e.g., adenovirus 2), bovine papilloma virus,fowl sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, andmost preferably simian virus 40 (SV40), and heterologous mammalianpromoters, such as actin promoter, immunoglobulin promoter, and heatshock promoter. Examples of enhancers that act on promoters to improvethe transcription include enhancers from known mammalian genes (i.e.,globin, elastase, albumin, α-fetoprotein, and insulin) and enhancersfrom eukaryotic cell viruses (e.g., SV40 late enhancer at replicationorigin, enhancer (bp 100-270), a polyoma late enhancer at replicationorigin, and polyoma enhancer and adenovirus enhancer can be used.

The expression vector can comprise a sequence necessary for terminationof transcription and stabilization of mRNA. Such sequences can beusually obtained from the 5′-non-translational region and occasionallyfrom the 3′-non-translational region of DNA or cDNA of an eukaryoticorganism or virus.

A gene can be introduced into a host by any method, and examples of suchmethod include the calcium ion method, electroporation, spheroplast, thelithium acetate method, the calcium phosphate method, and lipofection.Examples of methods for introducing a gene into an animal describedbelow include microinjection, a method involving the use ofelectroporation or lipofection to introduce a gene into an ES cell, andnuclear transplantation.

In the present invention, the antibodies of interest can be obtained byculturing transformants and sampling the antibodies of interest from theculture supernatant. Transformants are cultured using a medium suitablefor a host to be used via stationary culture, roller bottle culture, orthe like.

After culture, antibodies secreted extracellularly are purified using aculture liquid as such or by removing cells via centrifugation oranother means. Thereafter, general biochemical techniques via variouschromatography techniques for protein isolation/purification may beemployed solely or optionally in combination to isolate and purify thetarget antibodies from the culture product.

Furthermore, techniques for preparing a transgenic animal may beperformed to prepare animal hosts comprising genes for antibodies ofinterest incorporated into endogenous genes thereof, such as transgeniccattle, goats, sheep, or pigs. Thereafter, monoclonal antibodies derivedfrom such antibody genes can be obtained in a large quantity from themilk secreted from the transgenic animals (Wright, G., et al., 1991,Bio/Technology 9, 830-834).

Preferred methods for preparing the agonist antibodies to human Mpl ofthe present invention include, but are not limited to, methods usinggenetic recombination techniques as exemplified in the above-describedmeans for solving the problems.

3. DNA of the Present Invention

As described above, the present invention provides:

(1) DNA comprising a nucleotide sequence that encodes an amino acidsequence of a heavy chain variable region of an agonist antibody tohuman Mpl and that encodes an amino acid sequence selected from (a) to(d) below:

(a) the amino acid sequence as shown in SEQ ID NO: 2;

(b) the amino acid sequence as shown in SEQ ID NO: 4;

(c) the amino acid sequence as shown in SEQ ID NO: 6; and

(d) the amino acid sequence as shown in SEQ ID NO: 8; and

(2) DNA comprising a nucleotide sequence that encodes an amino acidsequence of a light chain variable region of an agonist antibody tohuman Mpl and that encodes an amino acid sequence selected from (a) to(h) below:

(a) the amino acid sequence as shown in SEQ ID NO: 3;

(b) the amino acid sequence as shown in SEQ ID NO: 5;

(c) the amino acid sequence as shown in SEQ ID NO: 7;

(d) the amino acid sequence as shown in SEQ ID NO: 9;

(e) an amino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues inthe framework region in the amino acid sequence as shown in SEQ ID NO:3;

(f) an amino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues inthe framework region in the amino acid sequence as shown in SEQ ID NO:5;

(g) an amino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues inthe framework region in the amino acid sequence(s) as shown in SEQ IDNO: 7; and

(h) an amino acid sequence comprising a deletion(s), substitution(s),addition(s), or insertion(s) of one or several amino acid residues inthe framework region in the amino acid sequence as shown in SEQ ID NO:9.

These DNAs can be used in the method for producing agonist antibodies tohuman Mpl of the present invention as described in section 2 above, andmore specifically in the method for producing antibodies using geneticrecombination techniques.

DNAs encoding the amino acid sequences (a) to (d) of the above describedvariable regions were obtained by extracting mRNA by conventionaltechniques as described later in Example 7 from hybridoma strainsobtained by the method for producing hybridomas that produce agonistantibodies to human Mpl as described above and by the 5′-RACE methodwith the use of primers prepared based on the amino acid sequences ofknown antibody constant regions. Plasmids comprising the DNAs encodingthe variable regions were deposited under the terms of the BudapestTreaty at the International Patent Organism Depositary of the NationalInstitute of Advanced Industrial Science and Technology (Tsukuba Central6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan) on Mar. 14, 2006.

TABLE 1 SEQ ID NO. (plasmid name) Accession Number Deposition Date 2FERM-BP-10559 Mar. 14, 2006 (Anti-hMpl 7-10_HV/pCR4) 4 FERM-BP-10553Mar. 14, 2006 (Anti-hMpl 4-49_HV/pCR4) 6 FERM-BP-10555 Mar. 14, 2006(Anti-hMpl 6-4-50_HV/pCR4) 8 FERM-BP-10557 Mar. 14, 2006 (Anti-hMpl6-5-2_HV/pCR4) 3 FERM-BP-10560 Mar. 14, 2006 (Anti-hMpl 7-10_LV/pCR4) 5FERM-BP-10554 Mar. 14, 2006 (Anti-hMpl 4-49_LV/pCR4) 7 FERM-BP-10556Mar. 14, 2006 (Anti-hMpl 6-4-50_LV/pCR4) 9 FERM-BP-10558 Mar. 14, 2006(Anti-hMpl 6-5-2_LV/pCR4)

The variable region of the light chain that constitutes the agonistantibody of the present invention comprises the amino acid sequence asshown in SEQ ID NO: 3, 5, 7, or 9 as a specific example. The frameworkregion of such amino acid sequence may comprise a deletion(s),substitution(s), addition(s), or insertion(s) of one or several aminoacid residues. Also, such amino acid sequence may comprise a sequencehaving at least 85%, 86%, 87%, 88% or 89%, preferably at least 90%, 92%,93% or 94%, and more preferably at least 95%, 96%, 97%, 98% or 99%identity with the sequence of the framework region. The term “frameworkregion” as used herein refers to a region excluding threecomplementarity-determining regions (CDRs), i.e., RASQGISS (A or T)LA(SEQ ID NO: 89) (amino acid positions 24-34), DASSLES (SEQ ID NO: 90)(amino acid positions 50-56), and QQFNSYP (L or Y or W)T (SEQ ID NO: 91)(amino acid positions 89-97) from the variable region, in the amino acidsequence as shown in SEQ ID NO: 3, 5, or 7. In case of the amino acidregion as shown in SEQ ID NO: 9, the term “framework region” refers to aregion excluding RASQSVSSSYLA (SEQ ID NO: 92) (amino acid positions24-35), DASSRAT (SEQ ID NO: 93) (amino acid positions 51-57), andQQYGSSPIT (SEQ ID NO: 94) (amino acid positions 90-98) from the variableregion. As demonstrated in Example 17 later, the mutant antibodies ofthe present invention can have an agonistic activity substantiallyequivalent to that of the unmutated antibody, even in the presence of anamino acid mutation in the framework region. More specifically, themutant antibody can have an ability to bind to the human thrombopoietinreceptor of a cell like FM3A-hMpl cell, thereby activating the receptor,and/or an ability to amplify UT-7/TPO cells.

An example of said mutation is substitution between conserved aminoacids. Conserved amino acids have a property, such as electric charge,structure, or polarity, similar to each other. Such conserved aminoacids can be classified into: basic amino acids (Arg, His, or Lys);acidic amino acids (Glu or Asp); nonpolar amino acids (Ala, Leu, Ile,Val, Gly, or Pro); polar amino acids (Ser, Thr, Cys, Met, Asn, or Gln);and aromatic amino acids (Phe, Tyr, or Trp), for example.

The sequence identity represents a percentage of matching residues in asequence alignment between two or more amino acid (or nucleotide)sequences with or without the introduction of a gap. The sequenceidentity generally is a percentage of the number of the same amino acids(or nucleotides) relative to the total number of amino acids (ornucleotides). The sequence identity can be determined by accessing thedatabank such as NCBI (U.S.A.) and utilizing known algorithms, such asBLAST or FASTA for sequence search, according to need.

Mutation can be introduced into DNA that encodes a mutation-free aminoacid sequence by, for example, site-directed mutagenesis or PCR (withthe use of mutation-containing primers). The method for introducingmutation is described in, for example, Sambrook et al., MolecularCloning A Laboratory Mannual, Cold Spring Harbor Laboratory Press, 1989.

The DNA of the present invention may further comprise a nucleotidesequence encoding a heavy chain or light chain constant region, inaddition to a variable region.

Modification of the heavy chain constant region as described in theabove section concerning the method for producing an antibody of thepresent invention can be achieved by well-known genetic engineeringtechniques based on the sequences of deposited DNA and of known humanantibody constant regions.

4. Pharmaceutical Use and Composition of Agonist Antibody to Human c-Mpl

The agonist antibody to human c-Mpl of the present invention has anability to bind and activate the c-MPL receptor and/or an ability tostimulate the production of platelets (i.e., platelet-producingactivity) and an ability to stimulate the production of plateletprogenitors (i.e., blood megakaryocyte-producing activity) in vivo andin vitro.

The human c-Mpl receptor is assumed to be expressed in hematopoieticstem cells as well as in megakaryocytes. It is reported thatadministration of PEG-rHuMGDF increased progenitor cells oferythroblasts or granulocytes/macrophage cells in the bone marrow ofnormal animals (Stem Cell, 14: 651-660, 1996). In mice into which thehuman umbilical cord blood had been transplanted, however,administration of PEG-rHuMGDF resulted in growth of progenitor cellsother than murine megakaryocytes, although growth of human progenitorcells was not observed. The number of progenitor cells of humanerythroblasts or granulocytes/macrophage cells was significantly largein the bone marrow of the agonist antibody to human c-Mpl (Example 14).This indicates that the agonist antibody to human c-Mpl introducessignals selectively in human cells to improve the viability of othercells as well as megakaryocytes.

Conditions to be treated by the pharmaceutical composition comprising,as an active ingredient, an agonist antibody to human c-Mpl according tothe present invention are generally accompanied with deficiency ofexisting megakaryocytes/platelets or with deficiency ofmegakaryocytes/platelets that is anticipated or predicted in the future(e.g., deficiency resulting from the planned surgery or plateletdonation). Such conditions are caused by temporary or permanentdeficiency of active Mpl ligands in vivo. Accordingly, the compositionof the present invention can be used for preventively or therapeuticallytreating thrombocytopenia of a patient who needs treatment of plateletdeficiency, i.e., thrombocytopenia. Further, the composition can be usedfor preventively or therapeutically treating pancytopenia of a patientwho needs treatment for recovery of blood cells after hematopoietic stemcells transplantation involving pancytopenia for a long period of time,such as bone marrow transplantation, umbilical cord bloodtransplantation, or peripheral blood stem cell transplantation.

Thrombocytopenia (deficiency of platelets) can be caused by variousreasons, including chemotherapy and other therapeutic methods with avariety of drugs, radiation therapy, surgery, accidental bleeding, andother concrete pathological conditions. Specific examples of typicalpathological conditions accompanying thrombocytopenia that can betreated by the present invention include: aplastic anemia; idiopathic orimmunothrombocytopenia (ITP), such as idiopathic thrombocytopenicpurpura associated with breast cancer; ITP associated with HIV andthrombotic thrombocytopenic purpura associated with HIV; metastatictumor causing thrombocytopenia; systemic erythematodes, such as aneonatal lupus syndrome associated with splenomegaly; Fanconi anemia;vitamin B12 deficiency; folic acid deficiency; May-Hegglin anomaly;Wiskott-Aldridge Syndrome; chronic hepatic failure; osteomyelodysplasiasyndrome associated with thrombocytopenia; paroxysmal nocturnalhemoglobinuria; acute profound thrombocytopenia following C7E3 Fab(Abciximab) therapy; isoimmune thrombocytopenia, such as maternalisoimmune thrombocytopenia; thrombocytopenia associated with anantiphospholipid antibody and thrombosis; autoimmune thrombocytopenia;immunothrombocytopenia induced by a drug, such as carboplatin-inducedthrombocytopenia or heparin-induced thrombocytopenia; fetalthrombocytopenia; thrombocytopenia during pregnancy; Hughes' syndrome;lupoid thrombocytopenia; accidental and/or massive hemorrhage;myeloproliferative disorders; thrombocytopenia in a patient with amalignant disease; thrombotic thrombocytopenic purpura, such asthrombotic microangiopathy that occurs in a cancer patient as thromboticthrombocytopenic purpura/hemolytic uremic syndrome; autoimmune hemolyticanemia; occult jejunal diverticulum perforation; pure red cell aplasia;autoimmune thrombocytopenia; epidemicity nephropathia;rifampicin-associated acute renal failure; Paris-Trousseauthrombocytopenia; neonatal alloimmune thrombocytopenia; paroxysmalnocturnal hemoglobinuria; hematologic changes in stomach cancer;hemolytic uremic syndromes in childhood; and hematologic manifestationsrelated to viral infection including hepatitis A virus andCMV-associated thrombocytopenia. Also, certain treatments for AIDSresult in thrombocytopenia (e.g., AZT). Certain wound healing disordersmight also benefit from an increase in platelet counts. Such diseasesinclude those accompanying other types of hematopenia, as well asthrombocytopenia.

The agonist antibodies of the present invention as the active ingredientcan be administered to treat the anticipated thrombocytopenia (e.g., dueto a surgery in the future) before platelets become necessary over theperiod of several hours to several days. In case of emergency (e.g.,accidental and massive hemorrhage), the agonist antibodies of thepresent invention can be administered with the blood or purifiedplatelets. Also, the agonist antibodies of the present invention as theactive ingredient can be administered to treat pancytopenia (e.g.,resulting from umbilical cord blood transplantation).

Examples of particularly preferable targets of treatment include (1)idiopathic thrombocytopenic purpura or thrombocytopenia accompanied byhepatic failure, and (2) thrombocytopenia and/or pancytopenia resultingfrom cancer chemotherapy, aplastic anemia, myelodysplasia syndrome(MDS), bone marrow transplantation, or umbilical cord bloodtransplantation.

The agonist antibody to human c-Mpl of the present invention can beuseful for maintaining the viability or storage life of platelets and/ormegakaryocytes and related cells. Accordingly, it is useful that aneffective amount of the agonist antibody is contained in a compositioncomprising such cells.

The pharmaceutical composition comprising, as an active ingredient, theagonist antibody to human c-Mpl according to the present invention maybe for administration for injection, oral, nasal, transdermal, or otherdosage forms, including, e.g., intravenous, intradermal, intramuscular,intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar,and intrapulmonary (e.g., aerosol drugs) administrations, orsubcutaneous injection (including depot administration for long-termrelease); and sublingual, anal, and vaginal administrations, or surgicalimplantation, e.g., embedded under the splenic capsule, in brain, or inthe cornea. The treatment may be conducted by a single dose or multipledoses over a given period of time. In general, the present inventionincludes pharmaceutical compositions comprising an effective amount ofthe agonist antibody to human c-Mpl of the present invention togetherwith pharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents of various buffer contents (e.g., Tris-HCl, acetate, orphosphate), pH and ionic strength; additives such as surfactants andsolubilizing agents (e.g., Tween 80 or Polysorbate 80), anti-oxidants(e.g., ascorbic acid or sodium metabisulfite), preservatives (e.g.,Thimersol or benzyl alcohol), and fillers (e.g., lactose or mannitol);particulate preparations of polymeric compounds (such as polylactic acidor polyglycolic acid) or liposomes, into which said active material hasbeen encapsulated. The pharmaceutical compositions may optionallyinclude still other pharmaceutically acceptable liquid, semisolid, orsolid diluents that serve as pharmaceutical vehicles, excipients, ormedia. Examples thereof include, but are not limited to, polyoxyethylenesorbitan monolaurate, magnesium stearate, methyl-andpropy-lhydroxybenzoate, starch, sucrose, dextrose, gum Arabic, calciumphosphate, mineral oil, cocoa butter, and oil of theobroma. Thecompositions may be prepared in liquid form, or may be in dried powder,such as lyophilized form. Implantable sustained release formulations andtransdermal formulations are also contemplated.

The dosage regimen involved in a method for treating the above-describedconditions will be determined by the attending physician, consideringvarious factors which modify the action of drugs, e.g. the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration, and other clinical factors.Generally, the dose should be in the range of 100 μg to 1 mg of theantibody or antibodies of the present invention per kilogram of bodyweight per day, preferably 10 to 100 μg/kg; and more preferably 1 to 10μg/kg, given in daily doses or in equivalent doses at longer or shorterintervals, e.g., every other day, twice weekly, weekly, or twice orthree times daily.

The pharmaceutical composition comprising, as an active ingredient, theagonist antibody to human c-Mpl according to the present invention maybe employed alone or in combination with other a cytokine(s), solubleMpl receptor, a hematopoietic factor(s), interleukin(s), or a growthfactor(s) in the treatment of disease states characterized by othersymptoms as well as platelet deficiencies. The agonist antibody to humanc-Mpl according to the present invention is expected to be useful intreating some types of thrombocytopenia in combination with a generalstimulator(s) of hematopoiesis, such as IL-3 or GM-CSF. Othermegakaryocytic stimulatory factors, such as meg-CSF, stem cell factor(SCF), leukemia inhibitory factor (LIF), oncostatin M (OSM), or othermolecules with megakaryocyte stimulating activity may also be employedwith Mpl ligand. Additional exemplary cytokines or hematopoietic factorsfor such co-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3,IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), M-CSF,SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO,interferon-alpha (IFN-alpha), consensus interferon, IFN-beta, IFN-gamma,IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, thrombopoietin (TPO), angiopoietins, for example Ang-1, Ang-2,Ang-3, Ang-4, Ang-Y, the human angiopoietin-like polypeptide, vascularendothelial growth factor (VEGF), angiogenin, bone morphogenicprotein-1, bone morphogenic protein-2, bone morphogenic protein-3, bonemorphogenic protein-4, bone morphogenic protein-5, bone morphogenicprotein-6, bone morphogenic protein-7, bone morphogenic protein-8, bonemorphogenic protein-9, bone morphogenic protein-10, bone morphogenicprotein-11, bone morphogenic protein-12, bone morphogenic protein-13,bone morphogenic protein-14, bone morphogenic protein-15, bonemorphogenic protein receptor IA, bone morphogenic protein receptor IB,brain derived neurotrophic factor, ciliary neurotrophic factor, ciliaryneurotrophic factor α, cytokine-induced neutrophil chemotactic factor 1,cytokine-induced neutrophil chemotactic factor 2α, cytokine-inducedneutrophil chemotactic factor 2β, β endothelial cell growth factor,endothelin 1, epidermal growth factor, epithelial-derived neutrophilattractant, fibroblast growth factor 4, fibroblast growth factor 5,fibroblast growth factor 6, fibroblast growth factor 7, fibroblastgrowth factor 8, fibroblast growth factor 8b, fibroblast growth factor8c, fibroblast growth factor 9, fibroblast growth factor 10, acidicfibroblast growth factor, basic fibroblast growth factor, glial cellline-derived neurotrophic factor receptor α1, glial cell line-derivedneurotrophic factor receptor α2, growth related protein, growth relatedprotein α, growth related protein β, growth related protein γ, heparinbinding epidermal growth factor, hepatocyte growth factor, hepatocytegrowth factor receptor, insulin-like growth factor I, insulin-likegrowth factor receptor, insulin-like growth factor II, insulin-likegrowth factor binding protein, keratinocyte growth factor, leukemiainhibitory factor, leukemia inhibitory factor receptor α, nerve growthfactor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4,placenta growth factor, placenta growth factor 2, platelet-derivedendothelial cell growth factor, platelet derived growth factor, plateletderived growth factor A chain, platelet derived growth factor AA,platelet derived growth factor AB, platelet derived growth factor Bchain, platelet derived growth factor BB, platelet derived growth factorreceptor α, platelet derived growth factor receptor β, pre-B cell growthstimulating factor, stem cell factor receptor, TNF, including TNF0,TNF1, and TNF2, transforming growth factor α, transforring growth factorβ, transforming growth factor β1, transforming growth factor β1.2,transforming growth factor β2, transforming growth factor β3,transforming growth factor β5, latent transforming growth factor β1binding protein I, transforming growth factor β binding protein II,transforming growth factor β binding protein III, tumor necrosis factorreceptor type I, tumor necrosis factor receptor type II, urokinase-typeplasminogen activator receptor, vascular endothelial growth factor, andchimeric proteins thereof.

Accordingly, administration of the pharmaceutical compositioncomprising, as an active ingredient, the agonist antibody to human c-Mplaccording to the present invention (for increasing the number of matureblood megakaryocytes) is expected to be a particularly effective meansfor stimulating platelet production. Such administration is alsoexpected to be an effective means for stimulating production ofhematopoietic stem cells. The aforementioned dose would be adjusted tocompensate for such additional components in the therapeuticcomposition. Progress of the treated patient can be monitored byconventional methods.

Hereafter, the present invention will be described in more detail withreference to Examples as described below; however, the technical scopeof the present invention is not limited to the examples.

EXAMPLES Example 1

Preparation of Antigen

1-1: Preparation of Human c-Mpl-Expressing Cells

When antigen protein-expressing cell lines are used for immunization, ingeneral, use of cell lines exhibiting a higher expression level is moreadvantageous for the preparation of antibodies. Various types of humanmegakaryocytic cell lines or forcibly-expressing cell lines are known ashuman c-Mpl-expressing cell lines; however, the c-Mpl expression levelsin such cell lines are as low as several thousands molecules per cell,and such cells are not suitable for antigens. When humanantibody-producing mice (KM mice™) were immunized with the FDCP-hMplexpressing cells that human c-Mpl gene had introduced into FDCP2, amurine hematopoietic cell line (see FEBS Lett. Oct. 21, 1996; 395 (2-3):228-34), in fact, an increase in the antibody titer was insufficient,and hMpl-specific human antibodies could not be obtained. When humanmegakaryocytic cell lines are used as the antigens, antibodies to othermembrane molecules are also induced. Accordingly, it is preferable thatmurine cell lines, and, if at all possible, cell lines expressing highlevels of human c-Mpl comprising human c-Mpl gene introduced inMHC-compatible host cell lines be used, in order to efficiently inducec-Mpl-specific antibodies. In order to prepare cells expressing highlevels of human c-Mpl (hMpl), hMpl expression vectors were prepared inthe following manner, and the resulting vectors were introduced into twotypes of murine cell lines (i.e., L929 and FM3A).

Further, a mutant receptor of hMpl that constantly transmits agonistsignals in a ligand-independent manner has been reported (a mutant inwhich Trp at position 508 has been substituted with Ser; Abe, M. et al.,Leukemia. August 2002; 16 (8): 1500-1506). Such mutant is deduced tohave a different conformation from that of a wild-type. An antibody thathas high affinity for a constantly active mutant may exhibit a potentagonist activity. Thus, an expression vector for the constantly activemutant (hereafter referred to as “hMpl-Ser”) was prepared, and then anexpression cell comprising the vector was also prepared for use inimmunization.

1) Preparation of Anti-Human c-Mpl (hMpl) Expression Vector

DNA of hump1-Pas12, which is a plasmid DNA carrying full-length cDNA ofhMpl (Bartley, T. D. et al., Cell, Jul. 1, 1994; 77 (7): 1117-1124 orMorita, H. et al., FEBS Lett. Oct. 21, 1996; 395 (2-3): 228-234), wasused as a template to conduct PCR for amplifying the entire hmpl codingregion. This region was amplified by PCR using the Mpl_F1 and Mpl_R2primers, which had been designed to comprise at the termini restrictionenzyme sites (i.e., EcoRI at the 5′ terminus and XbaI at the 3′terminus), and KOD-Plus-DNA polymerase (Toyobo, Japan). In the examplesset forth below, the reaction temperatures for PCR were adjusted usingthe GeneAmp® PCR System 9700 (Perkin Elmer Japan). The conditions forreaction temperatures were: heating at the initial temperature of 94° C.for 5 minutes; subsequently 30 cycles of 98° C. for 10 seconds and 68°C. for 3 minutes; and lastly heating at 72° C. for 7 minutes. Theamplified PCR fragment was recovered by ethanol precipitation, separatedby agarose gel electrophoresis, and then purified using the QIAquick gelextraction kit (Quiagen), which is a DNA purification kit using amembrane. The purified DNA fragment was subcloned into thepCR4Blunt-TOPO vector (Toyobo), and the nucleotide sequence of thecloned insert DNA in the plasmid was analyzed, and in this analysisM13-20FW and M13RV primers were used for DNA nucleotide sequencing. TheDNA nucleotide sequence of the inserted portion was analyzed, and aplasmid DNA, wherein the inserted portion was not different from thehMpl sequence (GenBank Accession No: M90102) and the primer portions hadthe same sequences as designed, was selected. Subsequently, the plasmidDNA comprising the hMpl nucleotide sequence was purified, digested withthe EcoRI and XbaI restriction enzymes, and subjected to agarose gelelectrophoresis to recover and purify a DNA fragment of a little smallerthan about 2 kb. Separately, the expression vector pEF6/Myc-His(Invitrogen), which has human EF promoter and blasticidin (Bsd)selection marker, and the pEGEP-N1 vector (BD Biosciences Clontech),which has CMV promoter and neomycin (Neo) selection marker, were alsodigested with the EcoRI and XbaI restriction enzymes, and treated withalkaline phosphatase (E. coli C75; TaKaRa Bio, Japan) fordephosphorylation. DNA was then recovered using agarose gelelectrophoresis and DNA purification kit. The DNA fragment of thepurified entire hMpl region was ligated to each expression vector DNAusing T4 DNA ligase, and the resultant was introduced into E. coli DH10Bto obtain a transformant. The DNA nucleotide sequences of plasmid DNA inthe insert DNA-containing transformant were analyzed, and pEF-MPL635 andpCMV-MPL635 into which the full-length hMpl cDNA had been inserted wereobtained.

(SEQ ID NO: 12) Mp1_F1: 5′-AGAGAGAGAG GAATTCGCCA CCATGCCCTC CTGGGCCCTCTT-3′ (SEQ ID NO: 13) Mp1_R2: 5′-AGAGAGAGAG CGGCCGCTCA AGGCTGCTGCCAATAGCTTA GTG-3′ (SEQ ID NO 14) M13-20FW: 5′-GTAAAACGACGGCCAGTG-3′ (SEQID NO: 15) M13RV: 5′-CAGGAAACAGCTATGAC-3′2) Preparation of Constantly Active Human c-Mpl (hMpl-Ser) ExpressionVector

An expression vector for the hMpl mutant, the intracellular signalactivation of which in a TPO-independent manner has been reported (i.e.,a mutant in which Trp at position 508 had been substituted with Ser;Abe, M. et al., Leukemia, August, 2002; 16 (8): 1500-1506), wasprepared. In order to change a codon encoding the amino acid residue atposition 508 (i.e., from TGG to TCG), the DNA of pEF-MPL635 was used asa template to perform site-directed mutagenesis using the GeneEditor™ invitro Site-Directed Mutagenesis System (Promega). Mut_MplSer508 was usedas a mutagenesis oligonucleotide (where the 5′-end has beenphosphorylated). The mutagenesis oligonucleotide of interest and theselection oligonucleotide included in the kit were annealed to thetemplate DNA to synthesize strands with mutation introduced. The mutantswere selected by utilizing the fact that only mutants are multiplied inthe presence of the GeneEditor™ Antibiotic Selection Mix. Morespecifically, after dsDNA template was incubated under alkalineconditions (0.2M NaOH, 0.2 mM EDTA (final concentrations)) at roomtemperature for 5 minutes, one-tenth volumes of 2M ammonium acetate (pH4.6) was added to neutralize the alkaline-denatured template DNAcontaining solution, followed by ethanol precipitation to recover thealkaline-denatured template DNA. To the alkaline-denatured template DNA,a mutagenesis oligonucleotide, a new antibiotic resistance acquiringselection oligonucleotide (where the 5′-end has been phosphorylated),and an annealing buffer accompanied with the kit were added, theresultant was heated at 75° C. for 5 minutes, and annealing was carriedout by slowly lowering the temperature to 37° C. Subsequently, thereaction was carried out with the use of Synthesis 10× buffer, T4 DNApolymerase, and T4 DNA ligase accompanied with the kit at 37° C. for 90minutes for synthesis and ligation of mutated strands. In the presenceof the GeneEditor™ Antibiotic Selection Mix, plasmid DNA was preparedfrom transformant E. coli strain which had been transformed and culturedin competent BMH 71-18 mutS cells, and the competent JM109 cells weresubsequently transformed with the plasmid DNA, which cells were theninoculated onto an LB plate comprising the GeneEditor™ AntibioticSelection Mix. The transformants emerged on the plate were cultured, andanalyzed for the DNA nucleotide sequence of the plasmid DNA, therebyobtaining pEF-MPL635-Ser vector that expressed hMpl in which the aminoacid residue at position 508 had been substituted (i.e., Trp to Ser).

(SEQ ID NO: 16) Mut_MplSer508: 5′-CTGCTGCTGC TGAGGTCGCA GTTTCCTGCACACTAC-3′3) Preparation of Full-Length Human c-Mpl Expressing L929 Cell

The prepared pEF-MPL635 vector (1 μg) was mixed with the lipofectaminereagent (purchased from Invitrogen) and the lipofectamine PLUS reagent(purchased from Invitrogen), then with serum-free Dulbecco's modifiedEagle medium (DMEM). The mixture was added to the L929 cells cultured ona 6-well plate (1.5×10⁵ cells/well), and the culture was conducted for 3hours in order to introduce the plasmid DNA into the cells. Theresulting cells were then cultured in a DMEM medium containing 10% fetalbovine serum (FBS) overnight. On the following day, 10 μg/ml blasticidin(purchased from Invitrogen) was added to the medium to selectdrug-resistant cells. Thereafter, c-Mpl-expressing cells were isolatedby the fluorescence activated cell sorting (FACS) method usinganti-c-Mpl antibodies, whereby a full-length human c-Mpl expressing L929cell line (hereafter referred to as “L929-hMpl”) was established. FACSwas carried out using the FACS-Vantage (Becton Dickinson). After theselection, the cells were cultured and maintained in DMEM mediumcontaining 5 μg/ml of blasticidin and 10% FBS.

4) Preparation of Full-Length Human c-Mpl Expressing FM3A Cells

In the same way as in 3) above, the pEF-MPL635 vector was introducedinto FM3A cells to establish the full-length human c-Mpl expressing FM3Acell line (hereafter referred to as “FM3A-hMpl”). The established cellswere cultured and maintained in Roswell Park Memorial Institute (RPMI)medium containing 5 μg/ml of blasticidin and 10% FBS.

5) Preparation of Constantly Active Human Mpl Expressing FM3A Cells

The pEF-MPL635-Ser vector as described above was introduced into FM3Acells in the same way as in 3) above to establish the hMpl-Serexpressing FM3A cell line (hereafter referred to as “FM3A-hMpl-Ser”).The cells were cultured and maintained in RPMI medium containing 5 μg/mlof blasticidin and 10% FBS.

1-2: Preparation of Solubilized Human c-Mpl Recombinant Protein

DNA encoding the solubilized human c-Mpl that lacks the transmembraneregion and the intracellular region of human c-Mpl and has the followingsequence was ligated to the expression vector pEAK8 (EdgeBioSystems),which was then introduced into the Hek293 cells with the aid of atransfectam reagent (available from Promega). After the stablyexpressing cells were selected, the culture supernatant thereof waspurified through an anti-Mpl antibody column to prepare solubilizedhuman c-Mpl recombinant protein (hereafter abbreviated to “solubleMpl-x” or “sMpl-x”).

(SEQ ID NO: 17) NH₂-MPSWALFMVTSCLLLAPQNLAQVSSQDVSLLASDSEPLKCFSRTFEDLTCFWDEEEAAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPDQEEVRLFFPLHLWVKNVFLNQTRTQRVLFVDSVGLPAPPSIIKAMGGSQPGELQISWEEPAPEISDFLRYELRYGPRDPKNSTGPTVIQLIATETCCPALQRPHSASALDQSPCAQPTMPWQDGPKQTSPSREASALTAEGGSCLISGLQPGNSYWLQLRSEPDGISLGGSWGSWSLPVTVDLPGDAVALGLQCFTLDLKNVTCQWQQQDHASSQGFFYHSRARCCPRDRYPIWENCEEEEKTNPGLQTPQFSRCHFKSRNDSIIHILVEVTTAPGTVHSYLGSPFWIHQAVRLPTPNLHWREISSGHLELEWQHPSSWAAQETCYQLRYTGEGHQDWKVLEPPLGARGGTLELRPRSRYRLQLRARLNGPTYQGPWSSWSDPTRVETATETAW-COOH

Example 2

Preparation of Monoclonal Antibody

The antibodies of the present invention were obtained by immunizing ahuman antibody-producing mouse (KM mouse™), which is capable ofproducing human antibodies via genetic modification, with an antigen,and preparing monoclonal antibodies. KM mouse is deficient in endogenousmouse immunoglobulin (Ig) heavy chain and mouse κ light chain, whilecarrying both a chromosome 14 fragment having human Ig heavy chain gene(SC20) and a human Igκ chain transgene (KCo5). Specifically, a KM mousehas the capacity for producing a human antibody and lacks the murine Igheavy chain and κ chain. This mouse is prepared by crossing between alineage A mouse having the human Ig heavy chain locus and a lineage Bmouse having the human Igκ chain transgene. The lineage A mouse ishomozygous for breakdowns of both endogenous Ig heavy chain and κ lightchain genes, which carries a progeny-transmittable chromosome 14fragment (SC20) lineage (see Tomizuka. et al., Proc. Natl. Acad. Sci.,U.S.A., 2000, Vol 97: 722). The lineage B mouse is homozygous fordeficiencies of both endogenous mouse Ig heavy chain and κ light chaingenes, which carries the human Igκ chain transgene (KCo5) (see Nat.Biotechnol., 1996, Vol 14: 845).

In the present example, monoclonal antibodies were prepared by knowntechniques (Introduction to Monoclonal Antibody Experiment Protocols,Ando, Tamie et al., Kodansha (Tokyo, Japan), 1991).

1) Immunization

As human c-Mpl immunogens, the L929-hMpl cells, FM3A-hMpl cells,constantly active c-Mpl expressing FM3A-hMpl-Ser cells, and sMpl-xrecombinant proteins as prepared in Example 1 were used. As the animalsto be immunized, the human antibody-producing mice producing the humanimmunoglobulin prepared in Example 2 were used, and immunization wascarried out in the following manner.

Immunization (Method 1):

L929-hMpl cells (5×10⁶ cells) prepared in Example 1 were mixed with theRibi adjuvant, and the mix was used to prime-immunize 9-week-old humanantibody-producing mice intraperitoneally. After priming, the mice wereimmunized with the same cells (2×10⁶ cells) and interleukin 6 (IL-6) (5μg) seven times at intervals of a week through their caudal veins, andfinally with the same cells via their caudal veins before removal of thespleen and lymph nodes from each mouse.

Immunization (Method 2):

The FM3A-hMpl-Ser cells (5×10⁶ cells) prepared in Example 1 wereirradiated with violet rays, the Ribi adjuvant was added thereto, andthe resultanting mix was used to prime-immunize 9-week-old humanantibody-producing mice intraperitoneally. After priming, the mice wereimmunized intraperitoneally with the same cells (5×10⁶ cells) seventimes at intervals of a week, and finally with the FM3A-hMpl cells(2×10⁶ cells) prepared in Example 1 and IL-6 (5 μg) via their caudalveins 3 days before removal of the spleen and lymph nodes from eachmouse.

Immunization (Method 3):

sMpl-x recombinant protein (10 μg) prepared in Example 1 was mixed withthe complete Freund's adjuvant (CFA), and the mix was used toprime-immunize 9-week-old human antibody-producing mice subcutaneously.The second to the fifth immunizations were carried out once a week bysubcutaneously immunizing the mice with a mix of the sMpl-x recombinantprotein (5 μg) and the incomplete Freund's adjuvant (IFA). The sixth tothe eighth immunizations were carried out by intraperitoneallyadministering the L929-hMpl cells (5×10⁶ cells). Finally, the sMpl-xrecombinant proteins (5 μg) and IL-6 (5 μg) were used to immunize themice via the caudal veins 3 days before removal of the spleen and thelymph nodes from each mouse.

2) Preparation of Hybridomas

The spleen and/or lymph nodes were surgically removed from the mouse 3days after the final immunization, placed in 10 ml of serum-free DMEMmedium containing 350 mg/ml of sodium bicarbonate, 50 units/ml ofpenicillin, and 50 μg/ml of streptomycin, and crushed using a spatula ona mesh (cell strainer, Falcon). The cell suspension that had passedthrough the mesh was subjected to centrifugation to precipitate thecells, which were then washed twice in serum-free DMEM medium andsuspended in a serum-free DMEM medium, and the cell counts weredetermined. Separately, the myeloma cell SP2/0 (ATCC No. CRL-1581) thathad been cultured in a 10% FCS-containing DMEM medium at 37° C. in thepresence of 5% carbon dioxide at a cell density no greater than 1×10⁸cells/ml, was also washed with serum-free DMEM medium, and thensuspended in a serum-free DMEM medium, and the cell counts weredetermined. The recovered cell suspension was mixed with a murinemyeloma cell suspension at a ratio of 5:1 in cell counts, the mixturewas centrifuged, and the supernatant was completely removed. To thispellet, 1 ml of 50% (w/v) polyethylene glycol 1500 (Boehringer Mannheim)as a fusion agent was slowly added with stirring with a tip of apipette, 1 ml of a serum-free DMEM medium preheated to 37° C. was slowlyadded in twice, and 7 ml of a serum-free DMEM medium was further added.After centrifugation, the supernatant was removed and the remainingfusion cells were subjected to screening for hybridomas of interestusing limited dilution as described below. Briefly, hybridomas wereselected by culturing in a DMEM medium containing 10% fetal calf serum(FCS) and hypoxanthine (H), aminopterin (A), and thymidine (T)(hereafter referred to as “HAT,” Sigma). Further, single-clone wasobtained by limited dilution using the DMEM medium comprising 10% FCSand HT (Sigma). Culture was conducted on a 96-well microtiter plate(Becton Dickinson). Hybridoma clones that produce anti-human c-Mpl humanmonoclonal antibodies were selected (or screened), and a humanmonoclonal antibody produced by each hybridoma was characterized by flowcytometry as described in Example 4 or cell proliferation assay usingthe UT7/TPO cells as described in Example 5. As a system for evaluatingagonist antibody activity, human Mpl may be expressed in a mouse cellline such as BaF3, followed by cell proliferation assay (Orita et al.,Blood, Jan. 15, 2005; 105 (2): 562-6). However, such reaction of thecells does not always reflect the reaction of human cells. Since theUT7/TPO is a human-derived cell line, its use for screening isconsidered to facilitate the selection of an antibody having strongeractivity on human cells.

As a result of the screening, as anti-human Mpl agonist antibodyproducing hybridomas, 4 clones, i.e., hybridoma 7-10 (obtained by Method1), hybridoma 4-49 (obtained by Method 2), and hybridomas 6-4-50 and6-5-2 (obtained by Method 3), were selected. A hybridoma that produced anon-agonist antibody, hybridoma 2-35 (obtained by Method 1) was selectedas a control.

Example 3

Preparation of Purified Antibody from Hybridoma Culture Supernatant

Anti-human c-Mpl monoclonal antibodies were purified from the hybridomaculture supernatants in the following manner. The antibody-containingculture supernatant was subjected to affinity purification using the rmpProtein A (Amersham Pharmacia Biotech), 0.8×40 cm column (Bio-Rad), PBSas an adsorption buffer, and 0.02 M glycine buffer (pH 3) as an elutionbuffer. The pH of elution fractions was adjusted at around 7.2 with theaddition of 1 M Tris (pH 9.0). The prepared antibody solutions weresubstituted with PBS using a dialysis membrane (10,000 cutoff, SpectrumLaboratories), and sterilized via filtration through a membrane filterMILLEX-GV (Millipore), pore size 0.22 μm, to obtain purified anti-humanc-Mpl monoclonal antibodies. The concentration of the purifiedantibodies was determined by measuring an absorbance at 280 nm and wascalculated by defining 1 mg/ml as being equal to 1.4 OD.

The anti-human c-Mpl monoclonal antibody-containing culture supernatantwas prepared in the following manner.

First, antibody-producing hybridomas were conditioned in an eRDF medium(Kyokuto Pharmaceutical Industrial) containing 10 ng/ml of recombinanthuman IL-6 (R&D Systems) and 10% low IgG fetal bovine serum (HyClone).The conditioned hybridomas were cryopreserved. Subsequently, some ofthem were conditioned in an eRDF medium (Kyokuto PharmaceuticalIndustrial) containing bovine insulin (5 μg/ml, Gibco-BRL), humantransferrin (5 μg/ml, Gibco-BRL), ethanolamine (0.01 mM, Sigma), sodiumselenite (2.5×10⁻⁵ mM, Sigma), 10 ng/ml recombinant human IL-6 (R&DSystems), and 1% low IgG fetal bovine serum (HyClone). Each of thehybridomas was cultured in a flask, and the culture supernatant wasrecovered when the % viability of the hybridoma reached 90%. Therecovered supernatant was applied to a 10-μm filter then a 0.2-μm filter(Gelman Science) to eliminate contaminants.

Example 4

Evaluation of Binding Activity of Anti-Human c-Mpl Antibodies by FlowCytometry

The binding activity of the anti-human c-Mpl antibodies was assayed byflow cytometry using the hybridoma culture supernatants or purifiedantibodies in the following manner. The FM3A-hMpl cells or human Mplexpressing FDCP2 cells (FDCP-hMpl) (FEBS, Lett., Oct. 21, 1996, 395(2-3): 228-34) were used.

Cells (4×10⁵ cells) were suspended in 50 μl of FACS staining medium (2%FBS, 0.1% NaN₃, 1 mM EDTA in PBS) per reaction, 50 μl of the hybridomaculture supernatant or purified human antibody solution (finalconcentration: 0.1-1 μg/ml) was added, and the reaction was carried outon ice for 30 minutes. After the washing with FACS staining medium, asecondary antibody, the R-phycoerythrin (RPE) labeled goat anti-humanIgγ F(ab′) antibody (Cat#2043-09, Southern Biotechnology), was added,and the reaction was carried out again on ice under the light-shieldedconditions for 30 minutes, followed by washing again. The cells weresuspended in a propidium iodide (PI)-containing FACS staining medium inorder to analying binding activities of the antibodies. The analysis wasmade using the FACS Calibur (Becton Dickinson).

FIG. 1 shows the results of flow cytometry using different purifiedantibodies. Each antibody was bound to FDCP-hMpl cell but not to theparent cell strain thereof, FDCP2 cell (“FDCP parent”). Thus, it wasdemonstrated that those antibodies each bound specifically to the humanMpl.

Example 5

Evaluation of Agonist Activity of Anti-Human c-Mpl Antibodies UsingUT7/TPO Cells

UT7/TPO cell proliferation assay was carried out using the hybridomasupernatants or purified antibodies to evaluate agonist activity. TheUT7/TPO cell was a TPO-dependent human megakaryocytic cell line (seeOzaki K et al., Blood, Dec. 15, 1998; 92 (12): 4652-62). In general, thecell was cultured and maintained in the Iscove's modified Dulbecco'smedium (IMDM) containing 10% FBS and 5 ng/ml of PEG-rHuMGDF. Cellproliferation assay was carried out in the following manner.

(1) The UT7/TPO cell culture was placed in a 50-ml tube and centrifugedto prepare a pellet of the cells (the centrifugation conditions: 1,500rpm, 5 min, 4° C.). The medium was removed, and the pellet was suspendedin a cytokine-free, 10% FBS-containing IMDM medium (hereafter referredto as a “proliferation assay medium”). The cells were recentrifuged andsuspended in a fresh proliferation assay medium. The centrifugation andsuspension was repeated once more.

(2) The cells suspended in the proliferation assay medium in (1) abovewere cultured at 37° C. in the presence of 5% CO₂ for 6 hours.

(3) After the culture, the cells were centrifuged to prepare a pellet,which was then suspended in the proliferation assay medium. In thiscase, the cell density was adjusted at 6×10⁵ cells/ml, and 50 μl of thecell suspension was plated in each well of a 96-well plate.

(4) Subsequently, 40 μl of the proliferation assay medium was added to10 μl of the hybridoma culture supernatant, and the resultant was addedto each well. When purified antibodies were used, the specimen was addedto 50 μl of the proliferation assay medium at a concentration of 2×final concentration, and the resultant was added to the wells.

(5) Culture was conducted at 37° C. in the presence of 5% CO₂ for 48hours.

(6) The WST-8 reagent (Dojindo Laboratories) was added at aconcentration of 10 μl/well, and culture was conducted for 2 hours.

(7) The absorbance in each well was measured using an absorptionmicroplate reader (Sunrise Rainbow; Tecan) (measurement wavelength: 450nm; reference wavelength: over 600 nm).

FIG. 2 shows the proliferation curves obtained by the UT7/TPO cellproliferation assay using the purified antibodies 7-10 (FIG. 2A), 4-49(FIG. 2B), 6-4-50 (FIG. 2C), and 6-5-2 (FIG. 2D). Table 2 below showsthe subclasses of the anti-human c-Mpl antibodies, and the intensitiesof activities (a 50% effective concentration (EC50) and a maximumactivity (Max), determined by the UT7/TPO cell proliferation assay),which were determined as results of the screening, along with theimmunization methods described in Example 2 by which the antibodies wereprepared.

TABLE 2 Sub- ST7/TPO UT7/TPO Immunization Hybridoma class (EC50) (Max)method 2-35 IgG1 − — 1 Non-agonist 7-10 IgG1 ++ >90% 1 4-49 IgG1 ++ >80%2 6-4-50 IgG1 + >80% 3 6-5-2 IgG1 + >50% 3 PEG-rHuMGDF — 0.001-0.01 nM100% — +: EC₅₀: 1-10 nM ++: EC₅₀: 0.1-1 nM

Example 6

Colony Assay

CFU-Mk colony formation assay was carried out using a human umbilicalcord blood-derived CD34+ cell, and the effect of the purified antibodieson the human primary cell were examined. Assay was carried out using theMegaCult™-C (Cat#04972, Stem Cell Technologies) in the following manner.

(1) The MegaCult™-C medium (0.85 ml) was added to 0.15 ml of the IMDMcontaining a specimen to bring the total volume of 1 ml.

(2) The CD34+ cells prepared from the human umbilical cord blood weresuspended in IMDM at a concentration of 1.1×10⁵ cells/ml, and 0.05 mlaliquots from the suspension were added to separate tubes containing themedium of (1) above.

(3) The tube containing the cells was vortexed, to which was 0.6 ml ofan ice-cooled collagen solution, and the mixture was then vortexedagain.

(4) The cell-specimen mixture of (1) to (3) above was added to each wellof the chamber slide in an amount of 0.75 ml.

(5) The chamber slide was placed in a 100-mm petri dish. In order toprevent the slide from drying, a 35-mm petri dish containing 3 ml ofpurified water was placed in the same 100-mm petri dish.

(6) The petri dish containing the chamber slide was left to stand in anincubator, and culture was conducted at 37° C. in the presence of 5% CO₂for 10 to 12 days.

(7) After the culture, the cells were fixed with a fixing solution(methanol:acetone=1:3).

(8) Immunostaining was carried out using an anti-human CD41 antibody todetect CFU-Mk colonies. The colony counts were counted microscopically,and the abilities of specimens to form CFU-Mk colonies were comparedwith one another.

FIG. 3 shows the results of colony assay. Colony formation was inducedby 7-10_IgG1 or 4-49_IgG1.

Example 7

Cloning and Sequencing of Antibody Gene

In order to prepare recombinant antibodies, antibody genes, specificallyhuman Igγ cDNA encoding heavy chain (H chain) and human Igκ cDNAencoding light chain (L chain), were isolated from the selectedhybridomas producing anti-human c-Mpl agonist antibodies, and sequencesthereof were determined.

1) Synthesis of cDNAs of Monoclonal Antibodies

In order to obtain DNA fragments comprising variable regions of thehuman antibody heavy chain and light chain expressed in hybridomas,cloning was carried out by the 5′-RACE (5′ rapid amplification of cDNAends) method using primers specific to constant regions of human Igγ andof human Igκ. Specifically, the cloning was carried out using the BDSMART RACE cDNA Amplification Kit (BD Biosciences Clontech) inaccordance with the attached instructions.

As the materials for cDNA synthesis, Isogen (Nippon Gene, Japan) for RNAextraction was added to hybridomas 7-10, 4-49, 6-4-50, and 6-5-2 cells,and total RNA was purified in accordance with the manufacturer'sinstructions. The 1st strand cDNA was prepared with the use of about 1μg of the purified total RNA as a template.

The 1st strand cDNA was synthesized in the following manner. Thereaction solution containing 1 μg/3 μl of total RNA, 1 μl of 5′ CDS, and1 μl of SMART Oligo was incubated at 70° C. for 2 minutes. Thereafter, 2μl of 5× buffer, 1 μl of DTT, 1 μl of DNTP mix, and 1 μl of PowerScriptReverse Transcriptase were added thereto, and the mixture was incubatedat 42° C. for 1.5 hours.

Further, 50 μl of tricine-EDTA buffer was added, and the mixture wasincubated at 72° C. for 7 minutes to obtain the 1 st strand cDNA.

2) Amplification of Heavy Chain Gene and Light Chain Gene by PCR andConfirmation of Nucleotide Sequences

2-1) Amplification of Heavy Chain and Light Chain Genes by PCR

In order to amplify cDNA for the human antibody gene, a 3′-primer havinga human antibody-specific sequence (specifically described below) and a5′-primer hybridizing specifically to a sequence added to the 5′ end ofcDNA synthesized using the BD SMART RACE cDNA Amplification Kit(Universal primer A mix) were used as a set of primers for PCR, and theKOD-Plus-DNA polymerase (Toyobo) was used as an enzyme for PCR toprepare a reaction solution having the composition shown below. Thissolution was used for PCR.

Sterile H₂O 28 μl cDNA 2.5 μl KOD-Plus-buffer (10×) 5 μl dNTPs Mix (2mM) 5 μl MgSO₄ (25 mM) 2 μl KOD-Plus- (1 unit/μl) 1 μl Universal primerA mix (UPM) (10×) 5 μl Gene specific primers (GSP) (10 μM) 1.5 μl Totalvolume 50 μl

The heavy chain gene was amplified using the UPM primer and the IgG1pprimer, which primers were included in the SMART RACE cDNA AmplificationKit. On the other hand, the light chain gene was amplified using a setof the UPM primer and the hk-2 primer.

(SEQ ID NO: 18) IgG1p primer: 5′-TCTTGTCCACCTTGGTGTTGCTGGGCTTGTG-3′ (SEQID NO: 19) hk-2: 5′-GTT GAA GCT CTT TGT GAC GGG CGA GC-3′

The reaction was carried out under the following temperature conditions:

A cycle of 94° C. for 30 seconds and 72° C. for 3 minutes was repeated 5times, a cycle of 94° C. for 30 seconds, 70° C. for 30 seconds, and 72°C. for 3 minutes was repeated 5 times, and a cycle of 94° C. for 30seconds, 68° C. for 30 seconds, and 72° C. for 3 minutes was repeated 25times.

Further, 98 μl of tricine-EDTA buffer was added to 2 μl of the abovereaction solution, 5 μl of the diluted solution was used as a template,and the second PCR (nested PCR) was carried out using primers set moreinward compared with the case of the first PCR. The composition of thePCR solution is shown below.

Sterile H₂O 30 μl First PCR reaction solution (50-fold diluted) 5 μlKOD-Plus-buffer (10×) 5 μl dNTPs Mix (2 mM) 5 μl MgSO₄ (25 mM) 2 μlKOD-Plus- (1 unit/μl) 1 μl Nested universal primer A (NUP; 10 μM) 1 μlGene specific primers (GSP) (10 μM) 1 μl Total volume 50 μl

When amplification of the heavy chain gene was carried out, the NUPMprimer (accompanied with the SMART RACE cDNA amplification kit; BDBiosciences Clontech) was used in combination with the hh2 primer (incase of hybridomas 4-49, 6-4-50, and 6-5-2) or the IgG2p_(—)134 primer(in case of hybridomas 7-10). When amplification of the light chain genewas carried out, the UPM primer and the hk-5 primer were used. Thereaction was carried out by heating at an initial temperature of 94° C.for 1 minute, followed by heating at 94° C. for 5 seconds, at 68° C. for10 seconds, and at 72° C. for 3 minutes for 20 cycles, followed byheating at 72° C. for 7 minutes.

2-2) Determination of Nucleotide Sequence of Antibody Gene

The PCR fragment of the heavy chain amplified by the above-describedmanner (hereafter referred to as “HV[C]”) is composed of the5′-untranslated region, the leader sequence (the secretion signalsequence), the variable region (HV), and part of the constant region([C]) of the heavy chain. Similarly, the PCR-amplified fragment of thelight chain (hereafter referred to as “LV [C]”) is composed of the5′-untranslated region, the leader sequence (the secretion signalsequence), the variable region (LV), and part of the constant region([C]) of the light chain. The term “leader sequence (secretion signal)”used herein refers to an amino acid sequence, which is required forsecretion of an antibody and is cleaved from a mature antibody protein.The HV[C] fragment and the LV[C] fragment are recovered from a PCRsolution by ethanol precipitation, separated by agarose gelelectrophoresis, and then purified with a DNA purification kit using amembrane, the QIAquick gel extraction kit (Qiagen). The purifiedamplified HV[C] fragment and the amplified LV[C] fragment were eachsubcloned into the pCR 4 Blunt-TOPO vector (Toyobo) of the Zero BluntTOPO PCR Cloning Kit (Invitrogen). The nucleotide sequence of DNAinserted into a plasmid of the obtained clone was analyzed. The M13-20FWand the M13RV primers were used in order to determine the nucleotidesequence of DNA.

(SEQ ID NO: 20) hk-5: 5′-AGG CAC ACA ACA GAG GCA GTT CCA GAT TTC-3′ (SEQID NO: 21) hh2 primer: 5′-GCT GGA GGG CAC GG TCA CCA CGC TG-3′ (SEQ IDNO: 22) IgG2p_134: 5′-TGCACGCCGC TGGTCAGGGC GCCTGAGTTC C-3′

The nucleotide sequences of DNAs encoding the heavy chain variableregion and the light chain variable region of the agonist antibody 7-10and the amino acid sequences of the heavy chain variable region and ofthe light chain variable region are shown below.

<Nucleic Acid Sequence of Heavy Chain of 7-10> (ATG Initiation Codon toDNA Sequence Encoding C-Terminal Amino Acid Residues of the VariableRegion)

(SEQ ID NO: 23) ATGGAGTTGGGACTGAGCTGGATTTTCCTTTTGGCTATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAAATCTATGGTTCGGGGAGTTCCGTTACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA<Amino Acid Sequence of Heavy Chain of 7-10> (Leader Sequence toVariable Region)

(The underlined amino acid residues compose a leader sequence as asecretion signal.)

(SEQ ID NO: 24) MELGLSWIFLLAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKNLWFGEFRYWYFD LWGRGTLVTV SS<Nucleic Acid Sequence of Light Chain of 7-10> (ATG Initiation Codon toDNA Sequence Encoding C-Terminal Amino Acid Residues of the VariableRegion)

(SEQ ID NO: 25) ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA<Amino Acid Sequence of Light Chain of 7-10>(Leader Sequence to VariableRegion)

(The underlined amino acid residues compose a leader sequence as asecretion signal.)

(SEQ ID NO: 26) MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK

The nucleotide sequences of DNAs encoding the heavy chain variableregion and the light chain variable region of the agonist antibody 4-49and the amino acid sequences of the heavy chain variable region and ofthe light chain variable region are shown below.

<Nucleic Acid Sequence of Heavy Chain of 4-49> (ATG Initiation Codon toDNA Sequence Encoding C-Terminal Amino Acid Residues of the VariableRegion)

(SEQ ID NO: 27) ATGGAGTTGGGACTGAGCTGGATTTTCCTTGTGGCTATTTTAAAAGGTGTCCAGTGTGAAGAGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCACCTTTGATGATTATGCCATGTACTGGGTCCGGCAAGTTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAACAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCGTTTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTATATTACTGTGCAAAAGCCCTATGGTTCGGGGAGTTCCCCCACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA<Amino Acid Sequence of Heavy Chain of 4-49> (Leader Sequence toVariable Region)

(The underlined amino acid residues compose a leader sequence as asecretion signal.)

(SEQ ID NO: 28) MELGLSWIFLVAILKGVQCEEQLVESGGGLVQPGRSLRLSCTASGFTFDDYAMYWVRQVPGKGLEWVSGISWNSGSIGYADSVKGRFTVSRDNAKNSLYLQMNSLRAEDTALYYCAKALWFGEFPHYYGMDVWGQGTTVTVSS<Nucleic Acid Sequence of Light Chain of 4-49> (ATG Initiation Codon toDNA Sequence Encoding C-Terminal Amino Acid Residues of the VariableRegion)

(SEQ ID NO: 29) ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTACTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGT<Amino Acid Sequence of Light Chain of 4-49> (Leader Sequence toVariable Region)

(The underlined amino acid residues compose a leader sequence as asecretion signal.)

(SEQ ID NO: 30) MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSTLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKR

The nucleotide sequences of DNAs encoding the heavy chain variableregion and the light chain variable region of the agonist antibody6-4-50 and the amino acid sequences of the heavy chain variable regionand of the light chain variable region are shown below.

<Nucleic Acid Sequence of Heavy Chain of 6-4-50> (ATG Initiation Codonto DNA Sequence Encoding C-Terminal Amino Acid Residues of the VariableRegion)

(SEQ ID NO: 31) ATGGAATTGGGACTGAGCTGGATTTTCCTTTTGGCTATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAACCTCTGGATTCACCTTTGATAATTATGCCATGTACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTGACATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAGGGATGCGGGGTTCGGGGAGTTCCACTACGGTCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA<Amino Acid Sequence of Heavy Chain of 6-4-50> (Leader Sequence toVariable Region)

(The underlined amino acid residues compose a leader sequence as asecretion signal.)

(SEQ ID NO: 32) MELGLSWIFLLAILKGVQCEVQLVESGGGLVQPGRSLRLSCATSGFTFDNYAMYWVRQAPGKGLEWVSGISWNSGDIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDAGFGEFHYGLDVWGQGTTVTVSS<Nucleic Acid Sequence of Light Chain of 6-4-50> (ATG Initiation Codonto DNA Sequence Encoding C-Terminal Amino Acid Residues of the VariableRegion)

(SEQ ID NO: 33) ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGT<Amino Acid Sequence of Light Chain of 6-4-50> (Leader Sequence toVariable Region)

(The underlined amino acid residues compose a leader sequence as asecretion signal.)

(SEQ ID NO: 34) MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKVPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPWTFGQGTKVEIKR

The nucleotide sequences of DNAs encoding the heavy chain variableregion and the light chain variable region of the agonist antibody 6-5-2and the amino acid sequences of the heavy chain variable region and ofthe light chain variable region are shown below.

<Nucleic Acid Sequence of Heavy Chain of 6-5-2> (ATG Initiation Codon toDNA Sequence Encoding C-Terminal Amino Acid Residues of the VariableRegion)

(SEQ ID NO: 35) ATGGAGTTGGGACTGAGCTGGATTTTCCTTTTGGCTATTTTAAAAGGTGTCCAGTGTGAAGTGCAACTGGTGGAGTGTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGTATAGGTTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAACCTATATGGTTCGGGGAGTGGGGAAACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA<Amino Acid Sequence of Heavy Chain of 6-5-2> (Leader Sequence toVariable Region)

(The underlined amino acid residues compose a leader sequence as asecretion signal.)

(SEQ ID NO: 36) MELGLSWIFLLAILKGVQCEVQLVECGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKPIWFGEWGNYYGMDVWGQGTTVTVSS<Amino Acid Sequence of Light Chain of 6-5-2> (ATG Initiation Codon toDNA Sequence Encoding C-Terminal Amino Acid Residues of the VariableRegion)

(SEQ ID NO: 37) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGT<Amino Acid Sequence of Light Chain of 6-5-2> (Leader Sequence toVariable Region)

(The underlined amino acid residues compose a leader sequence as asecretion signal.)

(SEQ ID NO: 38) METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKR

Example 8 Construction of Recombinant Antibody Expression Vector

The antibody variable region cloned from the hybridoma in theabove-described manner was incorporated into the human antibodyexpression vector, and a recombinant antibody expression vector havingvarious constant regions was prepared.

The human antibody expression vector, N5KG1-Val Lark (hereafterabbreviated as “N5KG1”) (IDEC Pharmaceuticals, see U.S. Pat. No.6,001,358), is a plasmid vector used for expressing a recombinantantibody in an animal cell. The structure of N5KG1 is shown in FIG. 4A.N5KG1 comprises two CMV promoters/enhancers and, downstream thereof,cloning sites for genes of heavy chain and light chain variable regions.Further, N5KG1 originally comprises, downstream of such cloning sites,gene sequences encoding the human heavy chain constant region (γ1) andthe human light chain constant region (κ). Any heavy chain and lightchain variable regions (including the leader sequence, or the secretionsignal sequence) may be incorporated in frame into the cloning site ofthe variable region of the vector, whereby an antibody comprising thelight chain variable region ligated to the constant region of the humanκ chain and the heavy chain variable region ligated to the constantregion of the human γ1 chain can be expressed. Accordingly, the animalcells into which the vector has been introduced produce the IgG1antibody in the culture medium.

Similarly, the expression vector N5KG4PE (IDEC Pharmaceuticals)comprises the heavy chain constant region of IgG4PE. IgG4PE is asequence with two mutations, Ser228Pro and Leu235Glu, introduced intoIgG4. Ser228Pro is a mutation that suppresses formation of a monomerresulting from an intramolecular crosslinking of IgG4 (i.e., an S—Sbond), and Leu235Glu is a mutation that reduces the activity ofantibody-dependent cellular cytotoxicity (ADCC).

The IgG1 constant region of N5KG1 was converted into IgG3 to prepareN5KG3.

In this example, expression vectors were prepared from N5KG1, N5KG3, andN5KG4PE by adding various modifications to the heavy chain constantregion (in particular, a hinge region).

At the outset, modification provided to the constant region in thisexample was substitution of subclasses between antibody domains. Theantibody heavy chain constant region has a domain structure ofCH1-hinge-CH2-CH3 from the N-terminal side. In this example, the heavychain constant region of each subclass was prepared by combining thesedomain units into the sequence of the subclass. For example, the heavychain constant region wherein the CH1 and hinge region sequences were ofhuman IgG3, and the CH2 and CH3 sequences were of human IgG1, wasprepared. An antibody having such heavy chain constant region wasdesignated subclasses in the order of CH1/ hinge/CH2/ CH3, which wasnamed IgG3/3/1/1 (hereafter referred to as, for example, IgG3311, byomitting “/”). Another heavy chain constant region wherein the hingeregion sequence was, for example, of human IgG3 and the CH1, CH2, andCH3 sequences were of human IgG4PE, was also prepared. An antibodyhaving such heavy chain constant region was named IgG4344.

Secondally, a modification of human IgG3 hinge region was prepared. Thehinge region of an antibody can be divided into an upper hinge and amiddle hinge. The term “upper hinge” refers to a sequence of from theresidue 216 to a residue at a more N-terminal side than the residue 226,where the residue numbers were based on the Kabat EU numbering system(Kabat et al., Sequences of Proteins of Immunological Interest, 5^(th)Ed., Public Health Service, National Institute of Health, Bethesda, Md.,1991). The term “middle hinge” refers to a sequence of from the residue226 to a residue at a more N-terminal side than the residue 231, wherethe residue numbers were based on the same system. The hinge region ofhuman IgG3 is composed of 12 amino acid residues for upper hinge and 50amino acid residues for middle hinge, wherein the middle hinge isdivided into 5 amino acids and 3 repeats of 15 amino acids (i.e.,5+15×3=50). In this example, a mutant wherein the repeat of the IgG3middle hinge sequence was shortened to one time was prepared. Such hingewas named G3h1, and an antibody having this type of a hinge was combinedwith a mutation of said domain units, and the resulting combination wasdesignated as IgGx3xxh1 (where x is arbitrary).

Also, a heavy chain constant region that lacks a repeat sequence of thelast half of the IgG3 middle hinge was prepared. Such hinge was namedG3uh (the abbreviation of the “upper hinge”), and denoted as IgGx3xxuh.

Further, mutations, L217S and R228P, were added to the G3uh hinge toprepare a heavy chain constant region. This mutation is intended tobring G3uh hinge to a position closer to the IgG4PE sequence. Theresultant was named G3uhm (the abbreviation of “upper hinge mutation”),and an antibody having the same was referred to as IgGx3xxuhm.

FIG. 4B shows the amino acid sequences of a naturally-occurring humanimmunoglobulin and hinge regions of IgG4PE, IgG4344, IgG4344h1,IgG4344uh, and IgG4344uhm.

In this example, a variable region of an anti-Mpl agonist antibody wasused to prepare an expression vector for an antibody having thefollowing constant regions:

IgG1, IgG4PE, IgG3311, IgG3331, IgG3344, IgG3344h1, IgG4344, IgG4344h1,IgG4344uh, and IgG4344uhm.

Hereafter, methods for preparing expression vectors are described.

1) Preparation of Anti-c-Mpl Antibody Expression Vector of Subclass IgG1

1-1) Preparation of Anti-Human c-Mpl Antibody 4-49_IgG1 and Antibody7-10_IgG1 Expression Vector

Expression vectors for antibodies 7-10 and 4-49 were prepared byinserting the heavy chain variable region, then the light chain variableregion into the N5KG1 vector.

FIG. 4C shows a process for preparing an expression vector. Plasmid DNAcomprising the HV[C] and LV[C] fragments of antibodies 7-10 and 4-49 (asdescribed in Example 7) were used as templates, and a set of primerscomprising, at the termini, restriction enzyme sites for ligation (SalIon the 5′-terminal side and NheI on the 3′-terminal side) was used toamplify the leader sequences and the variable regions of the heavy chainand the light chain via PCR using KOD-Plus-DNA polymerase. ThePCR-amplified leader sequences and the variable regions of the heavychain and the light chain were denoted as an HV fragment and an LVfragment.

The 7-10HV and 4-49HV fragments were inserted into N5KG1. Primers foramplifying an HV fragment are shown below.

7-10; 5′ primer for HV fragment: 40-3H5Sal (SEQ ID NO: 39) 5′-AGAGAGAGAGGTCGACCACC ATGGAGTTGG GACTGAGCTG GATTT-3′ 3′ primer for HV fragment:40-3H3Nhe (SEQ ID NO: 40) 5′-AGAGAGAGAG GCTAGCTGAG GAGACAGTGA CCAGGGTGCCA-3′ 4-49; 5′ primer for HV fragment: F24HSal (SEQ ID NO: 41)5′-AGAGAGAGAGGTCGACCACCATGGAGTTGGGACTGAGCTGGATTT- 3′ 3′ primer for HVfragment: C15H3Nhe (SEQ ID NO: 42)5′-AGAGAGAGAGGCTAGCTGAGGAGACGGTGACCGTGGT-3′

The reaction was carried out via heating at the initial temperature of94° C. for 1 minute, and 35 cycles of 94° C. for 5 seconds and 68° C.for 45 seconds, followed by heating at 72° C. for 7 minutes. Theamplified DNA fragment was digested with the restriction enzymes, SalIand NheI, and an about 430-bp DNA fragment was recovered via agarose gelelectrophoresis and then purified. Separately, the N5KG1 vector wassuccessively digested with the restriction enzymes, SalI and NheI, andtreated with alkaline phosphatase (E. coli C75) (Takara Shuzo, Japan)for dephosphorylation. Thereafter, an about 8.9-kb DNA fragment wasrecovered via agarose gel electrophoresis and with the use of a DNApurification kit. These two fragments were ligated to each other usingT4 DNA ligase and introduced into E. coli DH10B to obtain transformants.The nucleotide sequences of plasmid DNAs from the resultingtransformants were analyzed, and the plasmid DNAs, N5KG1_(—)7-10_Hv andN5KG1_(—)4-49_Hv, wherein the HV fragments have been inserted in framein a 5′ upstream region of the heavy chain constant region, wereobtained.

Subsequently, an LV fragment (consisting of the light chain leadersequence and the variable region) was inserted into a plasmid vectorwhich comprises the IV fragment inserted therein. Plasmid DNA comprisingan LV[C] fragment was used as a template, and primers comprising, at thetermini, restriction enzyme sites for ligation (BglII on the 5′-terminalside and BsiWI on the 3′-terminal side) were used to amplify the LVfragment by PCR. The primers used for amplifying an LV fragment are asshown below.

7-10; 5′-primer for LV fragment: 165-1B_L18Bgl (SEQ ID NO: 43)5′-AGAGAGAGAGATCTCTCACCATGGACATGAGGGTCCCCGCTC-3′ 3′-primer for LVfragment: 165_1B_L18_Bsi (SEQ ID NO: 44) 5′-AGAGAGAGAG CGTACGTTTGATCTCCACCT TGGTCCCTCC-3′ 4-49; 5′-primer for LV fragment: DNP_L1Bglp(SEQ ID NO: 45) 5′-AGAGAGAGAGATCTCTCACCATGAGGGTCCCCGCTCAGCTC-3′3′-primer for LV fragment: A27_R_N202 (SEQ ID NO: 46)5′-AGAGAGAGAGCGTACGTTTGATTTCCACCTTGGTCCCTTGGC-3′

The reaction was carried out via heating at the initial temperature of94° C. for 1 minute, followed by heating at 94° C. for 5 seconds and at68° C. for 45 seconds for 35 cycles, followed by heating at 72° C. for 7minutes. The amplified DNA fragment of the purified LV was subclonedinto the pCR4Blunt-TOPO vector (Toyobo, Japan). The nucleotide sequenceof the DNA inserted into a plasmid of the obtained clone was analyzed.In order to determine the DNA nucleotide sequence, M13-20FW and M13RVprimers were used. The DNA nucleotide sequence of the insert wasanalyzed, and plasmid DNAs (TOPO_(—)7-10_Lv and TOPO_(—)4-49_Lv), whichare not different from the template LV and which have the primersequences as designed, were selected. Subsequently, DNAs were digestedwith restriction enzymes BglII and BsiWI, and an about 400-bp DNAfragment was recovered via agarose gel electrophoresis and purified. Thepurified DNA fragment was ligated to an about 9.3-kb vector comprisingIV 7-10 or 4-49 digested with restriction enzymes BglII and BsiWI anddephosphorylated inserted therein, with the aid of T4 DNA ligase, andthe resultant was introduced into E. coli DH10B to obtain transformants.The DNA sequences or restriction enzyme cleavage patterns of thetransformants were analyzed to select clones comprising plasmid DNAs ofinterest. Further, the obtained antibody-expressing plasmid DNAs werepurified in a large amount in order to confirm that mutation did notoccur during the cloning process in the entire heavy chain region, theentire light chain region, and DNA nucleotide sequences located in thevicinity of the inserted region. Expression vectors, 7-10_IgG1 and4-49_IgG1, were designated as N5KG1_(—)7-10 and N5KG1_(—)4-49.

FIG. 4C shows a process for producing N5KG1_(—)7-10 and N5KG1_(—)4-49.

1-2) Preparation of Anti-Human c-Mpl Antibodies 6-4-50_IgG1 and6-5-2_IgG1 Expression Vector

The expression vectors for 6-4-50 and 6-5-2 were prepared by insertingthe light chain variable region, then the heavy chain variable regioninto the human antibody expression vectors.

Plasmid DNA comprising the LV[C] fragments of antibodies 6-4-50 and6-5-2 (as described in Example 7) were used as templates, and a set ofprimers comprising, at the termini, restriction enzyme sites forligation (BglII on the 5′-terminal side and BsiWI on the 3′-terminalside) was used to amplify DNA of the LV fragment (consisting of theleader sequence and the variable region of the light chain) via PCRusing KOD-Plus-DNA polymerase. The primers used are as shown below.

6-4-50; 5′-primer for LV fragment: 208LF (SEQ ID NO: 47)5′-AGAGAGAGAGATCTCTCACCATGGACATGAGGGTCCCCGCTCAGC- 3′ 3′-primer for LVfragment: 62LP3Bsi (SEQ ID NO: 48)5′-AGAGAGAGAGCGTACGTTTGATTTCCACCTTGGTCCCTTG-3′ 6-5-2; 5′-primer for LVfragment: A27_F (SEQ ID NO: 49)5′-AGAGAGAGAGATCTCTCACCATGGAAACCCCAGCGCAGCTTCTCTT C-3′ 3′-primer for LVfragment: 202LR (SEQ ID NO: 50)5′-AGAGAGAGAGCGTACGTTTAATCTCCAGTCGTGTCCCTTGGC-3′

The reaction was carried out via heating at the initial temperature of94° C. for 1 minute, followed by heating at 94° C. for 5 seconds and at68° C. for 45 seconds for 35 cycles, followed by heating at 72° C. for 7minutes. The amplified DNA fragment was digested with restrictionenzymes, BglII and BsiWI, and an about 400-bp DNA fragment was recoveredvia agarose gel electrophoresis and purified. Separately, the N5KG1vector was successively digested with the restriction enzymes, BglII andBsiWI, and treated with alkaline phosphatase (E. coli C75) (TakaraShuzo, Japan) for dephosphorylation. Thereafter, an about 8.9-kb DNAfragment was recovered via agarose gel electrophoresis and with the useof a DNA purification kit. These two fragments were ligated to eachother using T4 DNA ligase and introduced into E. coli DH10B to obtaintransformants. The nucleotide sequences of plasmid DNAs of the resultingtransformants comprising the insert DNA were analyzed, and plasmid DNAs,N5KG1_(—)6-4-50_Lv and N5KG1_(—)6-5-2_Lv, wherein the LV fragments havebeen inserted in frame in a 5′-upstream region coding for the humanantibody light chain constant region of N5KG1, were obtained.Subsequently, the HV fragment (consisting of the leader sequence and thevariable region of the heavy chain) was inserted into a plasmid vectorcomprising the LV fragment inserted therein. Plasmid DNA comprising theHV[C] fragment (as described in Example 7) was used as templates, and aset of primers comprising, at the termini, restriction enzyme sites forligation (SalI on the 5′-terminal side and NheI on the 3′-terminal side)was used to amplify the HV fragment via PCR. The primers used are shownbelow.

6-4-50;

5′-primer for HV fragment: 50-5-7Hsal (SEQ ID NO: 51) 5′-AGAGAGAGAGGTCGACCACC ATGGAATTGG GACTGAGCTG GATTTT-3′ 3′-primer for HV fragment:C15H3Nhe (SEQ ID NO: 52) 5′-AGAGAGAGAGGCTAGCTGAGGAGACGGTGACCGTGGT-3′6-5-2; 5′-primer for HV fragment: F24HSal (SEQ ID NO: 53)5′-AGAGAGAGAGGTCGACCACCATGGAGTTGGGACTGAGCTGGATTT- 3′ 3′-primer for HVfragment: L66H3Nhe (SEQ ID NO: 54)5′-AGAGAGAGAGGCTAGCTGAGGAGACGGTGACCGTGGTC-3′

The reaction was carried out via heating at the initial temperature of94° C. for 1 minute, followed by heating at 94° C. for 5 seconds and at68° C. for 45 seconds for 35 cycles, followed by heating at 72° C. for 7minutes. The amplified DNA fragment of the purified HV fragment wassubcloned into the pCR4Blunt-TOPO vector (Toyobo, Japan). The nucleotidesequence of the DNA inserted into the obtained plasmid clone wasanalyzed. To determine the DNA nucleotide sequence, M13-20FW and M13RVprimers were used. The DNA nucleotide sequence of the insert wasanalyzed, and plasmid DNAs (TOPO_(—)6-4-50_Hv and TOPO_(—)6-5-2_Hv),which are not different from the template HV and which have the primersequences as designed, were selected. Subsequently, the DNAs each weredigested with the restriction enzymes SalI and NheI, and about 430-bpDNA fragment was recovered via agarose gel electrophoresis and purified.Separately, the DNA fragment to be inserted was ligated to an about9.3-kb vector comprising an LV fragment of 6-4-50 or 6-5-2 digested withrestriction enzymes SalI and NheI and dephosphorylated, the resultantwas introduced into E. coli DH10B to obtain transformants, and cloneshaving the target plasmid DNA were selected from the transformants.Further, the resulting antibody-expressing plasmid DNAs were purified ina large amount in order to confirm that mutation did not occur duringthe cloning process in the entire heavy chain region, the entire lightchain region, and DNA nucleotide sequences located in the vicinity ofthe inserted region. The antibody expression vectors 6-4-50_IgG1 and6-5-2_IgG1 were designated as N5KG1_(—)6-4-50 and N5KG1_(—)6-5-2.

FIG. 4D shows a process for producing N5KG1_(—)6-4-50 andN5KG1_(—)6-5-2.

2) Preparation of Anti-Human c-Mpl Antibody of Subclass IgG4PE

The expression vector for the antibody of subclass IgG4PE was preparedusing the aforementioned N5KG4PE vector. Plasmid DNA of N5KG4PE wascleaved with the restriction enzymes NheI and BamHI, and a fragmentcontaining a heavy chain constant region was purified and then ligatedto the same restriction enzyme sites of the anti-cMpl antibodies,N5KG1_(—)7-10 and N5KG1_(—)4-49, to prepare N5KG4PE_(—)7-10 andN5KG4PE_(—)4-49.

3) Preparation of N5KG3

The expression vector N5KG3 for human IgG3 was prepared by substitutingthe IgG1 heavy chain constant region of N5KG1 with the IgG3 constantregion having the sequence shown below.

Amino Acid Sequence of IgG3 Constant Region

(SEQ ID NO: 55) STKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKLREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRYTQKSLSLSPGK*Nucleotide Sequence of IgG3 Constant Region

(SEQ ID NO: 56) CTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGTTTGGGCACCCAGACCTACACCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGCACCTGAACTCCTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA4) Preparation of IgG3311 Expression Vector

The IgG3311 expression vector was prepared by a reaction comprisingheating at 98° C. for 1 second, 60° C. for 30 seconds, and 72° C. for 30seconds using N5KG3 as a template and linkH and 13ch1-R primers, andthis reaction was repeated 15 times. Simultaneously, a reaction cycle of98° C. for 1 second, 60° C. for 30 seconds, and 72° C. for 30 secondsusing N5KG1 as a template and 13ch1 and linkH2 primers was repeated 15times. The amplified DNA fragment was purified using the PCRpurification kit, a purified DNA fragment was mixed with the equivalentamount of the other purified DNA fragment, a reaction cycle of 98° C.for 1 second, 60° C. for 30 seconds, and 72° C. for 30 seconds wasrepeated 5 times, and an additional 15 cycles of the reaction werecarried out with the addition of linkH and linkH2 primers. The amplifiedDNA fragment was cleaved with NheI and BamHI and substituted with theIgG1 constant region of the N5KG1 vector. This expression vector wasdesignated as N5KG3311.

(SEQ ID NO: 57) linkH: GGG TAC GTC CTC ACA TTC AGT GAT CAG (SEQ ID NO:58) 13ch1-R: GTC TTC GTG GCT CAC GTC CAC CAC CAC GCA (SEQ ID NO: 59)13ch1: TGC GTG GTG GTG GAC GTG AGC CAC GAA GAC (SEQ ID NO: 60) linkH2:TGA TCA TAC GTA GAT ATC ACG GC5) Preparation of IgG3331 Expression Vector

The IgG3311 expression vector was prepared by a reaction comprisingheating at 98° C. for 1 second, 60° C. for 30 seconds, and 72° C. for 30seconds using N5KG3 as a template and linkH and CH3consR primers, andthis reaction was repeated 15 times. Simultaneously, a reaction cycle of98° C. for 1 second, 60° C. for 30 seconds, and 72° C. for 30 secondsusing N5KG1 as a template and CH3cons and linkH2 primers was repeated 15times. The amplified DNA fragment was purified using the PCRpurification kit, a purified DNA fragment was mixed with an equivalentamount of the other purified DNA fragment, a reaction cycle of 98° C.for 1 second, 60° C. for 30 seconds, and 72° C. for 30 seconds wasrepeated 5 times, and an additional 15 cycles of the reaction werecarried out with the addition of linkH and linkH2 primers. The amplifiedDNA fragment was cleaved with NheI and BamHI and substituted with theIgG1 constant region of the N5KG1 vector. This expression vector wasdesignated as N5KG3311.

CH3consR: GGTGTACACCTGTGGCTCTCGGGGCTGCCC (SEQ ID NO: 61) CH3cons:GGGCAGCCCCGAGAGCCACAGGTGTACACC (SEQ ID NO: 62)

Hereafter, methods for preparing IgG3344, IgG3344h1, IgG4344, IgG4344h1,IgG4344uh, and IgG4344uhm are described. The constant regions thereofwere amplified by PCR, the amplified products were cloned to obtainplasmids. Such modified constant regions were substituted with the IgG1constant region of N5KG1_(—)7-10 or the like.

6) Preparation of IgG3344 and IgG3344h1 Constant Regions

The IgG3344 expression vector was prepared by PCR-based mutagenesis(site-directed mutagenesis by the overlap extension method) usingN5KG3331 and N5KG4PE as templates in the following manner.

PCR was carried out using N5KG3331 as a template and G3G4_P1_F andG3G4_P2_R as primers by heating at the initial temperature of 94° C. for1 minute, followed by heating at 94° C. for 15 seconds, at 55° C. for 10seconds, and at 68° C. for 1 minute for 35 cycles, followed by heatingat 72° C. for 7 minutes. Simultaneously, PCR was carried out using theaforementioned expression vector, N5KG4PE, as a template and G3G4_P3_Fand G3G4_P4_R as primers under the same conditions. The amplified DNAfragment was recovered by agarose gel electrophoresis and then purifiedusing the QIAquick gel extraction kit (Qiagen). Equivalent amounts ofthese purified DNA fragments were mixed with each other. The overlappedportions of these two DNA fragments were annealed and subjected to fivecycles of extension reactions comprising heating at the initialtemperature of 94° C. for 1 minute and 94° C. for 10 seconds, 55° C. for10 seconds, and 68° C. for 1.5 minutes. Thereafter, G3G4_P1_F andG3G4_P4_R primers were added to the reaction solution in order toamplify the full-length sequence, and a cycle of heating at 94° C. for 5seconds and 68° C. for 2 minutes was repeated 20 times, followed byheating at 72° C. for 7 minutes. G3G4_P1_F and G3G4_P4_R primerscomprise restriction enzyme sites (the NheI site in G3G4_P1_F and theBamHI site in G3G4_P4_R) in order to cleave the coding region of thehuman antibody constant region and substitute the same with the relevantregion of the antibody expression vector. The amplified PCR fragment wasrecovered by agarose gel electrophoresis and then purified with the useof the QIAquick gel extraction kit. The purified amplified fragment wassubcloned into the pCR 4 Blunt-TOPO vector of the Zero Blunt TOPO PCRcloning kit (Invitrogen), and the nucleotide sequence of DNA insertedinto a plasmid of the resulting clone was analyzed. Based on the resultsof nucleotide sequence analysis, a clone having the IgG3344 andIgG3344h1 constant regions was selected.

(SEQ ID NO: 63) G3G4_P1_F: 5′-AGAGAGGCTA GCACCAAGGG CCCATCG-3′ (SEQ IDNO: 64) G3G4_P2_R: 5′-GAACTCAGGT GCTGGGCACC TTGGGCACG-3′ (SEQ ID NO: 65)G3G4_P3_F: 5′-CCAAGGTGCC CAGCACCTGA GTTCGAGGGG GGA-3′ (SEQ ID NO: 66)G3G4_P4_R: 5′-AGAGAGGGAT CCTCATTTAC CCAGAGACAG GGA-3′7) Preparation of IgG4344 Constant Region

The IgG4344 expression vector was prepared by the reaction usingN5KG3331 as a template and G434_P5_F and G434_P6_R as primers comprisingheating at the initial temperature of 94° C. for 1 minute, followed byheating at 94° C. for 15 seconds, 55° C. for 10 seconds, and 68° C. for1 minute for 35 cycles, followed by heating at 72° C. for 7 minutes.Simultaneously, PCR was carried out under the same conditions usingN5KG4PE as a template and G434_P7_F and G3G4_P2_R as primers. Theamplified DNA fragments were recovered by agarose gel electrophoresisand then purified by the QIAquick gel extraction kit (Qiagen). These twopurified DNA fragments and the DNA fragment, which had been amplifiedand purified with the use of N5KG4PE as a template and G3G4_P3_F andG3G4_P4_R as primers (i.e., three types of DNA fragments) were subjectedto the overlap extension reaction. Specifically, the overlapped portionsof the three types of DNA fragments were annealed, heated at the initialtemperature of 94° C. for 1 minute, and extended by 5 cycles of 94° C.for 10 seconds, 55° C. for 10 seconds, and 68° C. for 1.5 minutes. Inorder to amplify the full-length sequence, G434_P5_F and G3G4_P4_Rprimers were added to the reaction solution, which was then subjected toheating at 94° C. for 5 seconds and at 68° C. for 2 minutes for 20cycles, followed by heating at 72° C. for 7 minutes. The amplified PCRfragment was purified by the QIAquick gel extraction kit and subclonedinto the pCR 4 Blunt-TOPO vector. The nucleotide sequence of the DNAinserted into the obtained plasmid clone was then analyzed. Based on theresults of the nucleotide sequence analysis, a clone having the IgG4344constant region was selected.

(SEQ ID NO: 67) G434_P5_F: 5′-AGAGAGGCTA GCACCAAGGG GCCATCC-3′ (SEQ IDNO: 68) G434_P6_R: 5′-GGTTTTGAGC TCAACTCTCT TGTCCACCTT GGTGTTGC-3′ (SEQID NO: 69) G434_P7_F: 5′-GTGGACAAGA GAGTTGAGCT CAAAACCCCA CTTGGTGACAC-3′8) Preparation of IgG4344h1 Constant Region

The IgG4344h1 expression vector was prepared by PCR using N5KG4344 as atemplate and G434_P5_F and G434_P6_R as primers comprising heating atthe initial temperature of 98° C. for 10 seconds, followed by heating at98° C. for 10 seconds, at 55° C. for 30 seconds, and at 72° C. for 1minute for 7 cycles, followed by heating at 98° C. for 10 seconds and at68° C. for 1 minute for 30 cycles, followed by heating at 72° C. for 7minutes. Pyrobest DNA Polymerase (Takara Bio) was used. Simultaneously,PCR was carried out under the same conditions using N5KG3344h1 as atemplate and G434_P7_F and G3G4_P4_R primers. The amplified DNAfragments were recovered by agarose gel electrophoresis and thenpurified by the QIAquick gel extraction kit (Qiagen). The equivalentamounts of these purified DNA fragments were mixed, the overlappedportions of the two DNA fragments were annealed, the annealed productwas extended by heating at the initial temperature of 98° C. for 10seconds, followed by heating at 98° C. for 10 seconds, at 55° C. for 30seconds, and at 72° C. for 1 minute for 7 cycles. G434_P5_F andG3G4_P4_R primers were added to the reaction solution in order toamplify the full-length sequence. Further, a cycle of 98° C. for 10seconds and 68° C. for 1 minute was repeated 30 times, followed byheating at 72° C. for 7 minutes. The amplified PCR fragment wasrecovered by agarose gel electrophoresis and then purified with the useof the QIAquick gel extraction kit. The purified amplified fragment wassubcloned into the pCR 4 Blunt-TOPO vector, and the nucleotide sequenceof DNA inserted into a plasmid of the resulting clone was analyzed.Based on the results of the nucleotide sequence analysis, a clone havingthe G4344h1 constant region was selected.

9) Preparation of IgG4344uh Constant Region

G4344uh was prepared by PCR using N5KG4344 as a template and G434_P5_Fand 17-1R as primers comprising heating at the initial temperature of98° C. for 10 seconds, followed by heating at 98° C. for 10 seconds, at50° C. for 30 seconds, and at 72° C. for 1 minutes for 5 cycles,followed by heating at 98° C. for 10 seconds, at 55° C. for 30 seconds,and at 72° C. for 1 minutes for 5 cycles, followed by heating at 98° C.for 10 seconds and at 68° C. for 1 minutes for 25 cycles, followed byheating at 72° C. for 7 minutes. Pyrobest DNA Polymerase (Takara Bio,Japan) was used. Simultaneously, PCR was carried out under the sameconditions using N5KG3344h1 as a template and 17-2F and G3G4_P4_R asprimers. The amplified DNA fragments were recovered by agarose gelelectrophoresis and then purified by the QIAquick gel extraction kit.Equivalent amounts of these purified DNA fragments were mixed, theoverlapped portions of the two DNA fragments were annealed, the annealedproduct was extended by heating at the initial temperature of 98° C. for10 seconds, followed by heating at 98° C. for 10 seconds and at 68° C.for 1 minute for 5 cycles, followed by heating at 98° C. for 10 seconds,at 55° C. for 30 seconds, and at 72° C. for 1 minute for 5 cycles.Thereafter, G434_P5_F and G3G4_P4_R primers were added to the reactionsolution in order to amplify the full-length sequence. A cycle ofheating at 94° C. for 30 seconds and 68° C. for 1 minute was repeated 30times, followed by heating at 72° C. for 7 minutes. The amplified PCRfragment was recovered by agarose gel electrophoresis and then purifiedwith the use of the QIAquick gel extraction kit. The purified amplifiedfragment was subcloned into the pCR 4 Blunt-TOPO vector, and thenucleotide sequence of DNA inserted into the obtained plasmid clone wasanalyzed. Based on the results of the nucleotide sequence analysis, aclone having the IgG4344uh constant region was selected.

(SEQ ID NO: 70) 17-1R: 5′-AGGTGCTGGG CACCGTGGGC ATGTGTGAGT TGT-3′ (SEQID NO: 71) 17-2F: 5′-CACACATGCC CACGGTGCCC AGCACCTGAG TTC-3′10) Preparation of IgG4344uhm Constant Region

The IgG4344uhm expression vector was prepared by PCR using N5KG4PE as atemplate and G434_P5_F and 17m-1R as primers comprising heating at theinitial temperature of 98° C. for 10 seconds, followed by heating at 98°C. for 10 seconds, at 50° C. for 30 seconds, and at 72° C. for 1 minutesfor 5 cycles, followed by heating at 98° C. for 10 seconds, at 55° C.for 30 seconds, and at 72° C. for 1 minutes for 5 cycles, followed byheating at 98° C. for 10 seconds and at 68° C. for 1 minutes for 25cycles, followed by heating at 72° C. for 7 minutes. Pyrobest DNAPolymerase was used. Simultaneously, PCR was carried out under the sameconditions using N5KG4PE as a template and 17m-2F and G3G4_P4_R asprimers. The amplified DNA fragments were recovered by agarose gelelectrophoresis and then purified by the QIAquick gel extraction kit.Equivalent amounts of these purified DNA fragments were mixed, theoverlapped portions of the two DNA fragments were annealed, the annealedproduct was extended by 7 cycles of 94° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 1 minute. Thereafter, G434_P5_F and G3G4_P4_Rprimers were added to the reaction solution in order to amplify thefull-length sequence, and a cycle of heating at 94° C. for 30 secondsand 68° C. for 1 minute was repeated 30 times, followed by heating at72° C. for 7 minutes. The amplified PCR fragment was recovered byagarose gel electrophoresis and then purified with the use of theQIAquick gel extraction kit. The purified amplified fragment wassubcloned into the pCR 4 Blunt-TOPO vector, and the nucleotide sequenceof DNA inserted into a plasmid of the resulting clone was analyzed.Based on the results of nucleotide sequence analysis, a clone having theIgG4344uhm constant region was selected.

(SEQ ID NO: 72) 17m-1R: 5′-TGTGTGAGTT GTGTCACCAA GTGGGGTTTT GGACTCAACTCTCTTGTCCA CCTTGGT-3′ (SEQ ID NO: 73) 17m-2F: 5′-ACCCCACTTG GTGACACAACTCACACATGC CCACCATGCC CAGCACCTGA GTTCGAG-3′

FIG. 4E shows the amino acid sequences of various modified heavy chains.

11) Preparation of Expression Vector for Antibody Comprising VariousModified Heavy Chain Constant Regions

Plasmid DNA having various modified heavy chain constant regions wascleaved with the restriction enzymes NheI and BamHI, and a sequence ofthe constant region was purified and separated. Subsequently, anti-humanc-Mpl antibody expression vectors, N5KG1_(—)7-10, N5KG1_(—)4-49,N5KG1_(—)6-4-50, and N5KG1_(—)6-5-2, were digested with the sameenzymes, and constant regions were substituted.

FIG. 4F shows the sequence of the heavy chain of 7-10_IgG4344uhm.

FIG. 4G shows the sequence of the light chain of 7-10_IgG4344uhm.

Example 9

Transient Expression of Anti-Human c-Mpl Antibodies in 293F Cells andPurification

DNA of the expression vector prepared in Example 8 was prepared usingthe EndoFree plasmid kit (Qiagen) and introduced into free 293 cells(Invitrogen Life Technologies) using the FreeStyle™ 293 expressionsystem (Invitrogen Life Technologies) to obtain an antibody-containingculture supernatant via transient expression. The culture supernatant(containing about 500 μm of IgG), which had been filtered through amembrane filter (pore diameter: 0.22 μm, Millipore), was applied to anaffinity column for antibody purification, i.e., HiTrap rProtein A FF(column volume: 1 ml) (Amersham Biosciences), washed with PBS (−),eluted with 20 mM citrate buffer (pH 3.4), and then recovered in tubescontaining 200 mM phosphate buffer (pH 7.0).

Example 10

Preparation of Recombinant Antibody

The constructed antibody expression vector was introduced into a hostcell to prepare an antibody expressing cell. As a host cell, a cell lineprepared by conditioning dhfr-deficient CHO DG44 cells (IDECPharmaceuticals Corporation) in a serum-free EX-CELL325PF(JRH) mediumwas used. The vector was introduced into the host cell viaelectroporation. The antibody expression vector (about 2 μg) waslinearized with the restriction enzyme AscI, the genes were introducedinto 4×10⁶ cells of CHO using the Bio-Rad electrophoreter at 350 V and500 μF, and the resultanting cells were seeded on a 96-well cultureplate. After the vector was introduced, G418 was added, and the culturewas continued. After the colony was observed, an antibody expressioncell line was selected. The selected CHO cell line was cultured inEX-CELL325-PF medium (JRH) (containing 2 mM glutamine, 100 units/ml ofpenicillin, 100 μg/ml of streptomycin, and hypoxanthine and thymidine(HT) supplement (1:100) (Invitrogen)) in the presence of 5% CO₂. Theculture supernatant was allowed to adsorb on the Mabselect Protein Acolumn (Amersham Pharmacia Biotech), washed with PBS, and then elutedwith a 20 mM citrate-Na buffer containing 50 mM NaCl (pH 3.4). Theeluate was neutralized with 50 mM Phosphate-Na (pH 7.0). The resultantwas diluted to about 1.5-fold with Milli-Q water in order to adjust theconductivity to 4.0 ms/cm or lower. Subsequently, the sample was appliedto and allowed to adsorb to a column comprising Q-Sepharose (Hitrap QHP) (Amersham Pharmacia Biotech) ligated to SP-Sepharose (HiTrap SP FF)(Amersham Pharmacia Biotech), washed with a 20 mM sodium phosphatebuffer (pH 5.0), and eluted with 1× PBS buffer. The prepared antibodysolution was sterilized via filtration through a 0.22 μm membranefilter, MILLEX-GV (Millipore). The concentration of the purifiedantibody was determined by measuring an absorbance at 280 nm andcalculating from values of the absorbance based on that 1.4 OD equals to1 mg/ml.

The activity of the modified recombinant antibody was determined by theUT7/TPO assay (Example 5). When compared with 4-49_IgG1, the activitiesof IgG3311 and IgG3331 were found to be enhanced (FIG. 5A), and theactivities of 7-10_IgG4344uhm and 4-49_IgG4344uhm were found to beequivalent to that of PEG-rHuMGDF.

Activities of various modified antibodies are summarized in Table 3.Activities of all agonist antibodies were found to be enhanced bymodification of constant regions. IgG1 and IgG4PE of antibodies 7-10 and4-49 had equivalent activities, and IgG4344uhm had a higher activitythan IgG4PE. In IgG4344uhm, the C-terminal amino acids at positions 4 to7 in the amino acid sequence of 7 amino acids at the upper hinge regionof IgG4PE have been substituted with the sequence at positions 4 to 12in the amino acid sequence of 12 amino acids at the upper hinge regionof IgG3 (see FIG. 4B). Thus, this region is considered to be importantin enhancing the activity.

TABLE 3 2-35 7-10 4-49 6-4-50 6-5-2 IgG1 — ++ ++ + + IgG4PE NT ++ ++ NTNT IgG3311 NT +++ +++ ++ ++ IgG3331 NT +++ +++ NT NT IgG3344 NT +++ NTNT ++ IgG3344h1 NT +++ NT NT NT IgG4344 NT +++ NT NT NT IgG4344h1 NT +++NT NT NT IgG4344uh NT +++ NT NT NT IgG4344uhm NT +++ +++ NT NT +: EC₅₀1-10 nM ++: EC₅₀ 0.1-1 nM +++: EC₅₀ 0.01-0.1 nM NT: not tested

Example 11

Signal Transduction by Agonist Antibodies

When TPO binds to a c-Mpl receptor, intracellular proteinphosphorylation takes place. Examples of known major pathways that areactivated by TPO include three pathways, i.e., Jak-STAT, Ras-MAPK, andPI3K-Akt. Phosphorylation signaling downstream of c-Mpl caused byagonist antibodies was analyzed by Western blotting using antibodiesspecific for phosphorylated proteins. The antibodies used are asfollows: anti-STAT5 (Cat#9352, Cell Signaling), anti-phospho-STAT5(Cat#9351L, Cell Signaling), anti-JAK2 (Cat#06-255, Upstate),anti-phospho-JAK2 (Cat#07-606, Upstate), anti-Erk1/2 (Cat#9272, CellSignaling,), anti-phospho-Erk1/2 (Cat#9271L, Cell Signaling), anti-Akt(Cat#9102, Cell Signaling), and anti-phospho-Akt (Cat#9101S, CellSignaling).

Assay was carried out using these antibodies in the following manner.

1) UT7/TPO cells were washed in a cytokine-free IMDM medium and culturedfor 6 hours.

2) After the culture, the cells were adjusted at 1×10⁶ cells/ml andseeded on a 6-well plate at 2 ml/well.

3) Agonist antibodies or PEG-rHuMGDF (as a positive control) were addedto the wells to stimulate the cells.

4) After the stimulation of from 5 minutes to 2 hours, the cells wererecovered and washed with ice-cooled PBS.

5) The cells were palletized by centrifugation, the supernatant wasdiscarded, the pellet was lysed in the PhosphoSafe™ extraction reagent(Cat#71296, Novagen), and centrifugation was carried out again, therebyrecovering the supernatant (the cell extract).

6) The cell extract obtained in 5) above was subjected to detection ofphosphorylated proteins via Western blotting.

The results are shown in FIG. 6. Phosphorylation observed by the agonistantibodies 7-10G4344uhm and 4-49G4344uhm was a phosphorylation occurringin the pathway similar to the TPO signal (FIG. 6A). Regarding theantibody 6-5-2, phosphorylation of Jak2 or STAT5 was not observed inIgG1, whereas it was observed in IgG3344 (FIG. 6B).

Example 12

Priming Effect on Human Platelets

Although TPO does not cause platelet aggregation, it has an effect ofpromoting the platelet aggregation caused by an aggregation inducingagent such as ADP (i.e., the priming effect). The priming effect of anagonist antibody on human platelets was tested by the followingprocedures.

1) The peripheral blood obtained from a healthy human volunteercontaining one tenth volumes of 3.1% (w/v) trisodium citrate as ananticoagulant was centrifuged at 140 g for 15 minutes to prepare theplatelet rich plasma (hereafter abbreviated as “PRP”).

2) Centrifugation was further carried out (2,500 g, 15 min) toprecipitate the blood cells, and the blood plasma was collected.

3) The platelet counts in PRP were measured and then adjusted at 3×10⁵cells/μL with the blood plasma.

4) A specimen was added to 100 μl of the platelet suspension prepared in3) above, and the mixture was incubated for 3 minutes with agitation.

5) 5 μl of 30 μM ADP (SIGMA) was added, and reduction in turbidityresulting from platelet aggregation was assayed on Hematracer 801 (MCMedical, Japan).

The results are shown in FIG. 7. In the presence of ADP added, thepriming effect of the agonist antibodies was observed. In the antibodiesalone (i.e., without ADP), platelet aggregation did not occur.

Example 13

Administration to Cynomolgus Monkeys

Agonist antibodies were administered to cynomolgus monkeys, in order toanalyze changes in platelet counts. To confirm the response ofindividuals to TPO, PEG-rHuMGDF was administered intravenously (10μg/kg) on the first day (day 0), the conditions were observed for 3weeks, and the purified agonist antibodies 7-10G4PE (individual A) and7-10G3344h1 (individual B) were administered intravenously at a dose of1 mg/kg, 21 days after the initial administration.

The results are shown in FIG. 8. Transient increase in platelet countscaused by PEG-rHuMGDF was observed in both individuals A and B. Theincrease in the platelet counts was observed in individual B after theadministration of the agonist antibody 7-10G3344h1. Also, serioustoxicity was not observed following administration of the antibody.

Example 14

Effect on Human Umbilical Cord Blood Transplantation Model

To confirm that the agonist antibodies prepared in Example 10 wouldpromote the formation of the human hematopoietic system in the humanumbilical cord blood transplantation model, an experiment was carriedout in the following manner.

The NOG (NOD/SCID/IL2-γR KO) mice (purchased from the Central Institutefor Experimental Animals (CIEA) (Kawasaki, Kanagawa, Japan)) wereirradiated with the radioactive rays as the graft pretreatment (twograys), and 1,000 to 10,000 human umbilical-cord-blood-derived CD34+cells were injected through the caudal veins.

The administration of the analyte was first carried out on the followingday of transplantation, thereafter once a week. The groups, analytes,and dosages are shown below. Each group consists of 6 mice, andadministration was carried out intraperitoneally. The body weight wasmeasured at the time of weekly administration.

<Groups, Analytes, and Dosages>

I: the number of transplants: 10,000, administration of PBS (as acontrol)

II: the number of transplants: 1,000, administration of PBS

III: the number of transplants: 10,000, administration of the antibody7-10G4344uhm, 100 μg/head/week

IV: the number of transplants: 1,000, administration of the antibody7-10G4344uhm, 100 μg/head/week

V: the number of transplants: 10,000, administration of TPO(PEG-rHuMGDF), 5 μg/head/week

VI: the number of transplants: 1,000, administration of TPO(PEG-rHuMGDF), 5 μg/head/week

On a day before the transplantation and 2, 4, and 6 weeks after thetransplantation, the peripheral blood was analyzed in the followingmanner.

<Procedure for Peripheral Blood Analysis>

The peripheral blood (about 70 μl) was taken from the orbital veins ofthe mice using capillary tubes.

The blood cell counts were determined using the KX-21 automated bloodcell analyzer (Sysmex).

In order to determine the chimeric rate of human platelets and ofleukocytes, the cells were stained with combinations of antibodies shownin A and B below and then analyzed by the FACS Calibur.

A (for platelet analysis): PE-labeled-anti-human CD41 antibody (R7058,Dako) plus FITC-labeled-anti-mouse CD41 antibody (#553848, BDPharmingen); and

B (for leukocyte analysis): APC-labeled-anti-human CD45 antibody(IM2473, Beckman Coulter, Inc.) plus FITC-labeled-anti-mouse CD45antibody (#553080, BD Pharmingen). At the time of analysis, fluorescentbeads for quantification (Flow-Count beads) were added to analyze agiven quantity of blood.

The chimeric rate of platelets or leukocytes was calculated by theformula: human cell counts/(human cell counts+mouse cell counts)×100(%).The total platelet counts in peripheral blood were multiplied by thechimeric rate to give human platelet counts.

Mice were sacrificed on the 6th week, and bone marrow cells were removedfrom the thigh bone. The bone marrow cells were subjected to colonyassay to determine the number of progenitor cells of human bloodmegakaryocytes (MK), erythrocytes (E), or granulocytes/macrophages (GM).Colony assay for detecting the megakaryocyte progenitor cells (CFU-Mk)was carried out by adding TPO (50 ng/ml) and SCF (100 ng/ml) in theculture. Culture was conducted at 37° C. in the presence of 5% CO₂ for12 days. The colonies were detected using the anti-human CD41 antibodiesin the same way as in Example 6. Colony assay for detecting progenitorcells of erythrocytes or granulocytes/macrophages was carried out usingthe Methocult system (Stem Cell Technologies) and adding EPO (4 IU/ml),SCF (100 ng/ml), IL-3 (20 ng/ml), and GM-CSF (10 ng/ml) in the culture.Culture was conducted at 37° C. in the presence of 5% CO₂ and 5% O₂ for14 days. After the culture, colony counts were determined under amicroscope.

FIGS. 9A, 9B, and 9C show the results of the above-mentionedexperiments.

The groups to which the antibodies had been administered exhibitedsignificantly higher peripheral blood human platelet counts than othergroups 6 weeks after the transplantation (FIG. 9A). This suggests thatthe agonist antibody 7-10G4344uhm promotes platelet recovery at the timeof umbilical cord blood transplantation. Further, the group to which theantibodies had been administered exhibited significantly higher humanerythrocytes and granulocytes/macrophage progenitor cells in the bonemarrow (FIG. 9B). Also, the CD45 chimeric rate, which indicates a ratioof human leukocytes to mouse leukocytes, was significantly high, showingclearly that human leukocytes increased in the groups to which theantibodies had been administered (FIG. 9C). This suggests that theantibody 7-10G4344uhm can promote survival of other lineages of cells aswell as megakaryocytes.

These findings suggest that the agonist antibodies act on cells presentupstream of the time point when blood cells are blanched into eachlineage of megakaryocytes, erythrocytes, and granulocytes/macrophages.Taking into consideration the finding that Mpl is expressed inhematopoietic stem cells, the agonist antibodies are highly likely topromote proliferation of hematopoietic stem cells.

In this experiment, however, the group to which TPO had beenadministered did not exhibit similar effects. This may be because TPOalso acts on mouse hematopoietic cells, the group to which TPO had beenadministered may thus undergo competition between human cells and mousecells in the bone marrow, and as a result, the effect on human cellsmight not been merely observed. The agonist antibodies of interest arecharacterized in that they selectively act on human Mpl. Accordingly,the effect of Mpl-mediated signals on the amplification of humanumbilical cord blood hematopoietic stem cells was demonstrated for thefirst time in vivo.

Example 15

Analysis of Antigenecity of Hinge-Modified Antibody

The agonist antibodies of the present invention are characterized inthat the activity is enhanced by modification of a hinge portion;although enhanced antigenecity resulting from the modification was anissue of concern. Based on the amino acid sequence of the hinge-modified7-10G4344uhm, the antigenecity was predicted by a computer simulation.

Foreign proteins that had been administered in vivo are incorporated inantigen presenting cells (APCs), such as dendritic cells or macrophagecells, and then degraded. Thereafter, peptides are presented by majorhistocompatibility complex (MHC) class II molecules (HLA class II,HLA-DR, DQ, and DP in the case of humans). Peptides presented by APCsare recognized by the T cell receptor (TCR), and T cells are activated.The activated T cells (helper T cells) activate B cells that expressantibodies that recognize the above antigens, and antibodies that reactwith foreign proteins are produced. In such a mechanism, the affinitybetween a peptide and MHC class II molecule is a major factor thatdefines the antigenecity. It is known that, because human MHC class IImolecule has many types (or polymorphism), the same peptide exhibitsremarkably different affinity depending on the types of the class IImolecule.

The amino acid sequences of different human antibodies having the7-10G4344uhm and IgG4PE constant regions were analyzed for affinity tovarious types of human HLA-DR, DQ, or DP molecule (using the database ofHLA molecules and the analytical algorithm, which were provided byAlgoNomics).

As a result, no new epitopes occurred by hinge modification. Thissuggests that the modified antibodies of the present invention would notraise a problem of antigenecity when they are used as a medicament.

Example 16

Administration of Antibodies to Human Mpl Transgenic Mice

The antibodies of the present invention do not cross-react with mouseMpl. In order to assay the efficacy, accordingly, transgenic (Tg) miceinto which human Mpl had been introduced as a foreign gene wereprepared, and the antibodies were administered to the Tg mice. At theoutset, a 5.5-kb promoter region of mouse Mpl was amplified by PCR andthen cloned into a pBluescript plasmid vector. Subsequently, thetranslated region and the 3′-untranslated region of human Mpl wereamplified by PCR, and then ligated to a site downstream of the mouse Mplpromoter. The resulting construct was injected into fertilized eggs ofthe C57BL/6 mouse, the resulting egg was transplanted into a surrogatemother, which was allowed to deliver offspring. Genomic DNA wasextracted from the tail 3 weeks after the delivery, and Tg mice wereselected by PCR. The obtained Tg mouse was allowed to cross with aC57BL/6 mouse in order to establish mouse lineages of interest.Expression of human Mpl in the bone marrow was analyzed.

As a result, Tg mouse lineages having a plurality of human Mplantibodies were obtained. In the bone marrows from lineage 39L,expression of human Mpl was observed via RT-PCR. The efficacy of theantibody was confirmed using mice of the lineage 39L.

The agonist antibody 7-10G4344uhm was administered to the mice in asingle dose (3 or 10 μg/ml), and changes in platelet counts in theperipheral blood were analyzed using the KX-21 automated blood cellanalyzer. The peripheral blood was taken from the orbital veins andassayed weekly. As a positive control, TPO (PEG-rHuMGDF) was used. Thegroups are shown below (6 mice per group).

Group I: 10 μg of 7-10G4344uhm was administered

Group II: 3 μg of 7-10G4344uhm was administered

Group III: 3 μg of TPO was administered

Group IV: PBS was administered

Group VI: 10 μg of wild-type 7-10G4344uhm was administered

The results are shown in FIG. 10. The platelet counts increased in thegroups to which the antibody had been administered and in the group towhich TPO had been administered. The platelet counts in the group towhich TPO had been administered returned to substantially the baseline 2weeks after the administration. In contrast, the platelet counts in thegroups to which the antibody had been administered remained increasedeven one month after the administration. This suggests that the agonistantibodies are very stable in the blood and are able to promotethrombopoiesis over a long period of time in a single dose. Thus, theagonist antibodies are particularly suitable for treatment of chronicthrombocytopenia.

Example 17

Evaluation of Activity of 7-10G4344uhm Light Chain Mutant

Mutation was introduced into the framework region of the light chainvariable region of the agonist antibody 7-10 (7-10VL), and effects onthe binding activity and the agonist activity were studied. The mutantlight chains are: the light chain of the agonist antibody 4-49 (V104L);and the mutant light chains of the agonist antibody 6-4-50 eachcomprising a single amino acid substitution (i.e., A43V and G100Q).These mutant light chains were combined with the heavy chain of7-10G4344uhm to prepare antibodies. As a result, their bindingactivities and agonistic activities were found to be equivalent to thoseof the original 7-10G4344uhm. When a mutation (Y94F) was introduced intothe complementarity determining region (CDR) in the light chain variableregion of the agonist antibody 7-10, however, the binding activity andthe agonistic activity were reduced to about one tenth of the originallevels. This indicates that the amino acid sequence of the light chainhas some degree of freedom.

The amino acid sequences of the light chains of various mutants and7-10VL are shown below. The sites of mutation are indicated in bold andunderlined.

7-10VL (SEQ ID NO: 3):AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRESGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGG GTKVEIK 7-10VL_V104L(4-49VL; SEQ ID NO: 85):AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGG GTK L EIK7-10VL_G100Q (6-4-50VL substitution product 1; SEQ ID NO: 86):AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFG Q GTKVEIK 7-10VL_A43V(6-4-50VL substitution product 2; SEQ ID NO: 87):AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGK V PKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGG GTKVEIK 7-10VL_Y94F(CDR substitution product; SEQ ID NO: 88):AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNS F PLTFGG GTKVEIKAnalysis of Binding Activity:

The concentrations of antibodies were prepared at 1, 0.1, and 0.01μg/ml, and flow cytometry was carried out using FM3A-hMpl cells. Theexperiment was carried out in accordance with the method described inExample 4. The anti-DNP (dinitrophenol) antibody (subclass IgG4; humanantibody) was used as a control. The light chain mutant antibodiesexhibited the binding activity equivalent to that of the antibody7-10G4344uhm (FIG. 11).

Analysis of Agonist Activity:

Cell proliferation assay using UT-7/TPO cells was carried out inaccordance with the method described in Example 5. The light chainmutant antibodies exhibited the agonistic activity equivalent to that ofthe antibody 7-10G4344uhm (FIG. 12).

INDUSTRIAL APPLICABILITY

The present invention provides anti-human cMpl agonist human antibodiesthat can be used as various therapeutic agents for thrombocytopenia. Thepresent invention also provides antibody constant regions that can beused as other agonist antibodies and that can provide satisfactorysafety and pharmacological effects.

The present invention provides agonist antibodies to human c-Mpl thatcan activate the human thrombopoietin receptor (c-Mpl) in the form of awhole antibody. Such agonist antibodies can be used as varioustherapeutic agents for thrombocytopenia, and it can be expected toremarkably contribute to the medical industry.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

Sequence Listing Free Text

-   SEQ ID NO: 11: Hinge domain mutant, UH2G3uhm-   SEQ ID NOs: 12 to 16: Primers-   SEQ ID NOs: 18 to 22: Primers-   SEQ ID NOs: 39 to 54: Primers-   SEQ ID NOs: 57 to 73: Primers-   SEQ ID NO: 74: G3344h1-   SEQ ID NO: 75: G3344-   SEQ ID NO: 76: G4344-   SEQ ID NO: 77: G4344h1-   SEQ ID NO: 78: G4344uh-   SEQ ID NO: 79: G4344uhm-   SEQ ID NO: 80: G4PE-   SEQ ID NO: 81: 7-10G4344uhm H chain-   SEQ ID NO: 82: 7-10G4344uhm H chain-   SEQ ID NO: 83: 7-10G4344uhm L chain-   SEQ ID NO: 84: 7-10G4344uhm L chain-   SEQ ID NO: 85: 7-10VL_V104L (mutant)-   SEQ ID NO: 86: 7-10VL_G100Q (mutant)-   SEQ ID NO: 87: 7-10VL_A43V (mutant)

1. An agonist antibody to a human thrombopoietin receptor (c-Mpl),wherein the agonist antibody comprises: (A) antibody constant regionscomprising amino acid sequences selected from the following: (1) aminoacid sequences of heavy chain and light chain constant regions of ahuman antibody, or (2) an amino acid sequence of a heavy chain constantregion with a heavy chain constant region domain substituted betweenhuman antibody subclasses, and an amino acid sequence of a light chainconstant region of a human antibody, wherein, the heavy chain constantregion of (A)(1) or (A)(2) comprises: i) an upper hinge portion havingthe amino acid sequence of SEQ ID NO: 10; ii) a middle hinge portionhaving the amino acid sequence selected from the group consisting of:(a) amino acids 18 to 67 of SEQ ID NO: 101; (b) amino acids 18 to 37 ofSEQ ID NO: 102; and (c) amino acids 18 to 22 of SEQ ID NO: 103; and iii)a CH1 Region having the amino acid sequence of amino acids 1 to 5 of SEQID NO: 99; and iv) a CH2 region having the amino acid sequence of (a)amino acids 18 to 25 of SEQ ID NO: 100, or (b) amino acids 18 to 25 ofSEQ ID NO: 99, or the heavy chain constant region of (A)(1) or (A)(2)comprises: i) an upper hinge portion having the amino acid sequence ofSEQ ID NO: 11; ii) a middle hinge portion having the amino acid sequenceof amino acids 18 to 22 of SEQ ID NO: 104; and iii) a CH1 Region havingthe amino acid sequence of amino acids 1 to 5 of SEQ ID NO: 99; and iv)a CH2 region having the amino acid sequence of (a) amino acids 18 to 25of SEQ ID NO: 100, or (b) amino acids 18 to 25 of SEQ ID NO: 99; and (B)antibody variable regions capable of binding to and activating a humanthrombopoietin receptor; wherein the agonist antibody has the followingproperties: (a) that the antibody induces colony formation at aconcentration of 10,000 ng/ml or lower as determined by the CFU-MKcolony formation assay using human umbilical cord blood derived CD34+cells; and (b) that the antibody has a maximal activity at least 50%higher than that of PEG-rHuMGDF, which comprises the amino acid sequenceas shown in SEQ ID NO: 1 having the following structure and has beenpegylated at the N terminus, and a 50% effective concentration (EC50) of100 nM or less in the cell proliferation assay using UT7/TPO cells: (SEQID NO: 1) PEG-NH-SPAPPACDLRVLSKLLRDSHVLHSRLSQCPEVHPLPTPVLLPAVDFSLGEWKTQMEETKAQDILGAVTLLLEGVMAARGQLGPTCLSSLLGQLSGQVRLLLGALQSLLGTQLPPQGRTTAHKDPNAIFLSFQHLLRGKVRFLMLVGGSTLCVRRAPPTTAVPS-COOH.


2. The antibody according to claim 1 having the properties (a) and (b):(a) that the antibody induces colony formation at a concentration of1,000 ng/ml or lower as determined by the CFU-MK colony formation assayusing human umbilical cord blood derived CD34+ cells; and (b) that theantibody has a maximal activity at least 70% higher than that ofPEG-rHuMGDF and an EC 50 of 10 nM or less in the cell proliferationassay using UT7/TPO cell.
 3. The antibody according to claim 1 havingthe properties (a) and (b): (a) that the antibody induces colonyformation at a concentration of 100 ng/ml or lower as determined by theCFU-MK colony formation assay using human umbilical-cord-blood-derivedCD34+ cells; and (b) that the antibody has a maximal activity at least90% higher than that of PEG-rHuMGDF and an EC 50 of 1 nM or less in thecell proliferation assay using UT7/TPO cell.
 4. The antibody accordingto claim 1, wherein the heavy chain and light chain variable regions areselected from the group consisting of following (1) to (8): (1) a heavychain variable region comprising the amino acid sequence as shown in SEQID NO: 2 and a light chain variable region comprising the amino acidsequence as shown in SEQ ID NO: 3; (2) a heavy chain variable regioncomprising the amino acid sequence as shown in SEQ ID NO: 4 and a lightchain variable region comprising the amino acid sequence as shown in SEQID NO: 5; (3) a heavy chain variable region comprising the amino acidsequence as shown in SEQ ID NO: 6 and a light chain variable regioncomprising the amino acid sequence as shown in SEQ ID NO: 7; (4) a heavychain variable region comprising the amino acid sequence as shown in SEQID NO: 8 and a light chain variable region comprising the amino acidsequence as shown in SEQ ID NO: 9; (5) a heavy chain variable regioncomprising the amino acid sequence as shown in SEQ ID NO: 2, and a lightchain variable region comprising an amino acid sequence comprising adeletion(s), substitution(s), addition(s), or insertion(s) of one orseveral amino acid residues of the framework region in the amino acidsequence as shown in SEQ ID NO: 3; (6) a heavy chain variable regioncomprising the amino acid sequence as shown in SEQ ID NO: 4, and a lightchain variable region comprising an amino acid sequence comprising adeletion(s), substitution(s), addition(s), or insertion(s) of one orseveral amino acid residues of the framework region in the amino acidsequence as shown in SEQ ID NO: 5; (7) a heavy chain variable regioncomprising the amino acid sequence as shown in SEQ ID NO: 6, and a lightchain variable region comprising an amino acid sequence comprising adeletion(s), substitution(s), addition(s), or insertion(s) of one orseveral amino acid residues of the framework region in the amino acidsequence as shown in SEQ ID NO: 7; and (8) a heavy chain variable regioncomprising the amino acid sequence as shown in SEQ ID NO: 8, and a lightchain variable region comprising an amino acid sequence comprising adeletion(s), substitution(s), addition(s), or insertion(s) of one orseveral amino acid residues of the framework region in the amino acidsequence as shown in SEQ ID NO:
 9. 5. The antibody according to claim 1,wherein the agonist antibody to human c-Mpl is a human antibody.
 6. Theantibody according to claim 5, wherein the antibody is selected from thegroup consisting of the following antibodies (1) to (8): (1) an antibodycomprising a heavy chain comprising the amino acid sequence as shown inSEQ ID NO: 2 and a light chain comprising the amino acid sequence asshown in SEQ ID NO: 3; (2) an antibody comprising a heavy chaincomprising the amino acid sequence as shown in SEQ ID NO: 4 and a lightchain comprising the amino acid sequence as shown in SEQ ID NO: 5; (3)an antibody comprising a heavy chain comprising the amino acid sequenceas shown in SEQ ID NO: 6 and a light chain comprising the amino acidsequence as shown in SEQ ID NO: 7; (4) an antibody comprising a heavychain comprising the amino acid sequence as shown in SEQ ID NO: 8 and alight chain comprising the amino acid sequence as shown in SEQ ID NO: 9;(5) an antibody comprising a heavy chain comprising the amino acidsequence as shown in SEQ ID NO: 2, and a light chain comprising an aminoacid sequence comprising a deletion(s), substitution(s), addition(s), orinsertion(s) of one or several amino acid residues of the frameworkregion in the amino acid sequence as shown in SEQ ID NO: 3; (6) anantibody having a heavy chain comprising the amino acid sequence asshown in SEQ ID NO: 4, and a light chain comprising an amino acidsequence comprising a deletion(s), substitution(s), addition(s), orinsertion(s) of one or several amino acid residues of the frameworkregion in the amino acid sequence as shown in SEQ ID NO: 5; (7) anantibody having a heavy chain comprising the amino acid sequence asshown in SEQ ID NO: 6, and a light chain comprising an amino acidsequence comprising a deletion(s), substitution(s), addition(s), orinsertion(s) of one or several amino acid residues of the frameworkregion in the amino acid sequence as shown in SEQ ID NO: 7; and (8) anantibody having a heavy chain comprising the amino acid sequence asshown in SEQ ID NO: 8, and a light chain comprising an amino acidsequence comprising a deletion(s), substitution(s), addition(s), orinsertion(s) of one or several amino acid residues of the frameworkregion in the amino acid sequence as shown in SEQ ID NO:
 9. 7. Apharmaceutical composition comprising, as an active ingredient, anantibody according to claim
 1. 8. A pharmaceutical composition forincreasing platelets comprising, as an active ingredient, an antibodyaccording to claim
 1. 9. The pharmaceutical composition for increasingplatelets according to claim 8, which is used for promoting plateletrecovery when bone marrow transplantation or umbilical cord bloodtransplantation is carried out.
 10. A therapeutic composition forthrombocytopenia comprising, as an active ingredient, an antibodyaccording to claim
 1. 11. The therapeutic composition forthrombocytopenia according to claim 10, wherein the thrombocytopenia isa disease selected from the group consisting of the following diseases(1) to (6): (1) idiopathic thrombocytopenic purpura (ITP); (2)thrombocytopenia caused after cancer chemotherapy; (3) aplastic anemia;(4) osteomyelodysplasia syndrome (MDS); (5) thrombocytopeniaattributable to hepatic diseases; and (6) thrombocytopenia caused afterbone marrow transplantation or umbilical cord blood transplantation. 12.A pharmaceutical composition for increasing blood cells used forpromoting blood cell recovery after hematopoietic stem celltransplantation, comprising, as an active ingredient, the antibodyaccording to claim 1.